Viral delivery of rna utilizing self-cleaving ribozymes and crispr-based applications thereof

ABSTRACT

The present disclosure relates to viral delivery of RNA utilizing self-cleaving ribozymes and applications of such, including but not limited to CRISPR-Cas related applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/311,887, filed Dec. 20, 2018, which is a U.S. national stage under 35U.S.C. § 371 of PCT International Patent Application No.PCT/US2017/038780, filed Jun. 22, 2017, which claims priority to U.S.Provisional Patent Application No. 62/353,452, filed Jun. 22, 2016, thecontents of each of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under grant number R21AI115226 awarded by the National Institute of Health (NIH). The UnitedStates government has certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention generally relates to viral delivery of RNAutilizing self-cleaving ribozymes and CRISPR-based applications thereof.

BACKGROUND OF THE INVENTION

Gene therapies broadly involve the delivery of nucleic acid polymers(e.g. RNA or DNA) into a cell in order to treat an underlying disease orcondition. One such gene therapy which has gained much attention overthe last several years is CRISPR/Cas9. CRISPR/Cas9 technology promisesto revolutionize genomics and modern medicine by allowing for theintroduction of point-level mutations into a host genome, e.g. allowingthe host genome to be cut at a desired location and then allowing genesto be removed or added. The CRISPR/Cas9 system generally relies ondelivery of a specific nuclease, typically Cas9, into a cell, which isguided by guide RNA (“gRNA”) to the appropriate section of the genomefor cutting. Known viral vector systems for delivery of CRISPR/Cas9,e.g. lentivirus and adeno-associated virus (AAV), have successfullymodified cells both ex vivo and in vivo; however, the DNA-basedreplication of these viruses carries the risk of unwanted integrationinto the host genome and thus genotoxicity or oncogenesis. Despite muchattention to this problem and innovations such as the use ofintegration-defective lentivirus, undesirable integration remains acarefully monitored risk that may affect the success of future genetherapy trials. These drawbacks are not simply unique to CRISPR/Cas9.Accordingly, there is an urgent need for improved viral vector deliverysystems, including for delivery of CRISPR-based technology.

SUMMARY OF THE INVENTION

The present disclosure relates to viral delivery of RNA utilizingself-cleaving ribozymes inserted into the RNA viral genome that areadjacent the target RNA to be delivered into the host cell, andparticularly the use of such in CRISPR-based technology. Accordingly, insome embodiments, the present disclosure is directed to a nucleic acid.In some embodiments, the nucleic acid comprises a genome sequence of asingle-stranded RNA (ssRNA) virus. In some embodiments, the genomesequence comprises antisense RNA. In some embodiments, the nucleic acidcomprises an antigenome sequence. In some embodiments, the antigenomesequence is complementary to the genome sequence. In some embodiments,the antigenome sequence comprises sense RNA. In some embodiments, theantigenome sequence comprises a first region. In some embodiments, thegenome sequence comprises a first region. In some embodiments, the firstregion comprises (i) a target segment and (ii) a first segmentcomprising a first self-cleaving ribozyme. In some embodiments, thefirst region further comprises (iii) a second segment encoding a secondself-cleaving ribozyme. In some embodiments, the target segment isadjacent to the first segment. In some embodiments, the target segmentis immediately upstream of the first segment. In some embodiments, thetarget segment is immediately downstream of the first segment. In someembodiments, the target segment is immediately upstream of the secondsegment. In some embodiments, the target segment is immediatelydownstream of the second segment. In some embodiments, the targetsegment is immediately upstream of a self-cleaving ribozyme. In someembodiments, the target segment is immediately downstream of aself-cleaving ribozyme. In some embodiments, the target segment isflanked by the first segment and the second segment. In someembodiments, the first self-cleaving ribozyme is a 5′ self-cleavingribozyme. In other embodiments, the first self-cleaving ribozyme is a 3′self-cleaving ribozyme. In some embodiments, the second self-cleavingribozyme is a 5′ self-cleaving ribozyme. In other embodiments, thesecond self-cleaving ribozyme is a 3′ self-cleaving ribozyme. In someembodiments, the target segment is flanked by a 5′ self-cleavingribozyme and a 3′ self-cleaving ribozyme. In some embodiments, the firstregion comprises an RNA expression cassette.

In some embodiments, the 5′ self-cleaving ribozyme is a hammerheadribozyme. In some embodiments, the 3′ self-cleaving ribozyme is ahammerhead ribozyme. In some embodiments, both the 5′ self-cleavingribozyme and the 3′ self-cleaving ribozyme are hammerhead ribozymes. Insome embodiments, neither the 5′ self-cleaving ribozyme and the 3′self-cleaving ribozyme are hammerhead ribozymes. In some embodiments,the 5′ self-cleaving ribozyme includes SEQ ID NO: 2. In someembodiments, SEQ ID NO: 2 has conservative substitutions. In someembodiments, the 3′ self-cleaving ribozyme includes SEQ ID NO: 3. Insome embodiments, SEQ ID NO: 3 has conservative substitutions. In someembodiments, the 3′ self-cleaving ribozyme is a hepatitis delta virus(HDV) ribozyme. In some embodiments, the 5′ self-cleaving ribozyme is ahammerhead ribozyme and the 3′ self-cleaving ribozyme is a hepatitisdelta virus (HDV) ribozyme. In some embodiments, the 5′ self-cleavingribozyme is a twister ribozyme. In some embodiments, the 3′self-cleaving ribozyme is a twister ribozyme. In some embodiments, boththe 5′ self-cleaving ribozyme and the 3′ self-cleaving ribozyme aretwister ribozymes. In some embodiments, the 5′ self-cleaving ribozyme isa twister sister ribozyme. In some embodiments, the 3′ self-cleavingribozyme is a twister sister ribozyme. In some embodiments, both the 5′self-cleaving ribozyme and the 3′ self-cleaving ribozyme are twistersister ribozymes. In some embodiments, the 5′ self-cleaving ribozyme isa pistol ribozyme. In some embodiments, the 3′ self-cleaving ribozyme isa pistol ribozyme. In some embodiments, both the 5′ self-cleavingribozyme and the 3′ self-cleaving ribozyme are pistol ribozymes. In someembodiments, the 5′ self-cleaving ribozyme is a hatchet ribozyme. Insome embodiments, the 3′ self-cleaving ribozyme is a hatchet ribozyme.In some embodiments, both the 5′ self-cleaving ribozyme and the 3′self-cleaving ribozyme are hatchet ribozymes. In some embodiments, the5′ self-cleaving ribozyme is a hairpin ribozyme. In some embodiments,the 3′ self-cleaving ribozyme is a hairpin ribozyme. In someembodiments, both the 5′ self-cleaving ribozyme and the 3′ self-cleavingribozyme are hairpin ribozymes.

In some embodiments, the antigenome sequence comprises a second region.In some embodiments, the genome sequence comprises a second region. Insome embodiments, the first region is upstream of the second region. Insome embodiments, the first region is downstream of the second region.In some embodiments, the second region comprises an expression cassette.

In some embodiments, the second region comprises a third segmentencoding a nuclease. In some embodiments, the nuclease comprises aCRISPR-associated protein (“Cas”). In some embodiments, the nucleasecomprises Cas9. In some embodiments, the nuclease comprises Cpf1. Insome embodiments, the nuclease comprises a Cas9-like protein or aCas9-like synthetic protein. In some embodiments, the nuclease comprisesa Cfp1-like protein or a Cfp1-like synthetic protein. In someembodiments, the nuclease comprises a C2c1 protein, C2c2 protein, C2c3protein, or variants and modifications thereof. In some embodiments, thenuclease comprises Class 2 CRISPR-associated nuclease. In someembodiments, the nuclease comprises a Class 2 Type II CRISPR-associatednuclease. In some embodiments, the nuclease comprises a Class 2 Type VCRISPR-associated nuclease. In some embodiments, the second regionfurther comprises a ribosomal skipping sequence. In some embodiments,the ribosomal skipping sequence is a P2A ribosomal skipping sequence. Insome embodiments, the second region comprises a fourth segment encodinga reporter molecule. In some embodiments, the reporter molecule includesa protein. In some embodiments, the reporter molecule is greenfluorescent protein (GFP). In some embodiments, the reporter molecule isred fluoresecent protein (RFP). In some embodiments, the reportermolecule is mCherry. In some embodiments, the target segment comprisestarget RNA. In some embodiments, the target segment comprises guide RNA(gRNA). In some embodiments, the gRNA has a scaffold sequence and atargeting sequence. In some embodiments, the target segment furthercomprises trans-activating crRNA (tracrRNA).

In some embodiments, the antigenome sequence comprises a third region.In some embodiments, the genome sequence comprises a third region. Insome embodiments, the third region is upstream of the first region. Insome embodiments, the third region is downstream of the first region. Insome embodiments, the third region is upstream of the second region. Insome embodiments, the third region is downstream of the second region.In some embodiments, the third region is upstream of the first regionand downstream of the second region. In some embodiments, the thirdregion is downstream of the first region and upstream of the secondregion. In some embodiments, the third region is flanked by the firstregion and the second region. In some embodiments, the third regioncomprises a fifth segment. In some embodiments, the fifth segmentcomprises a P gene. In some embodiments, the P gene comprises a mutant Pgene. In some embodiments, the mutant P gene has one or more of thefollowing mutations: D433A, R434A, K437A, and combinations thereof.

In some embodiments, the antigenome sequence comprises a fourth region.In some embodiments, the genome sequence comprises a fourth region. Insome embodiments, the fourth region is upstream of the first region. Insome embodiments, the fourth region is downstream of the first region.In some embodiments, the fourth region is upstream of the second region.In some embodiments, the fourth region is downstream of the secondregion. In some embodiments, the fourth region is upstream of the firstregion and downstream of the second region. In some embodiments, thefourth region is downstream of the first region and upstream of thesecond region. In some embodiments, the fourth region is upstream of thefirst region, second region, and third region. In some embodiments, thefourth region is downstream of the first region, second region, andthird region. In some embodiments, the fourth region is downstream ofthe first region, second region, and upstream of the third region. Insome embodiments, the fourth region is upstream of the first region,second region, and downstream of the third region. In some embodiments,the fourth region is upstream of the first region, third region, anddownstream of the second region. In some embodiments, the fourth regionis downstream of the first region, third region, and upstream of thesecond region. In some embodiments, the third region comprises a sixthsegment. In some embodiments, the sixth segment comprises a L gene. Insome embodiments, the L gene comprises a mutant L gene. In someembodiments, the mutant L gene has one or more of the followingmutations: N1197S, L15581, K1795E, and combinations thereof.

In some embodiments, the first region is heterologous. In someembodiments, the second region is heterologous. In some embodiments, thethird region is heterologous. In some embodiments, the fourth region isheterologous. In some embodiments, the first region and the secondregion are heterologous. In some embodiments, the first region and thethird region are heterologous. In some embodiments, the first region andthe fourth region are heterologous. In some embodiments, the secondregion and the third region are heterologous. In some embodiments, thesecond region and the fourth region are heterologous. In someembodiments, the first region, second region and the third region areheterologous. In some embodiments, the first region, second region andthe fourth region are heterologous. In some embodiments, the firstregion, second region, third region and the fourth region areheterologous. In some embodiments, the target segment is heterologous.In some embodiments, the first segment is heterologous. In someembodiments, the second segment is heterologous. In some embodiments,the third segment is heterologous. In some embodiments, the fourthsegment is heterologous. In some embodiments, the fifth segment isheterologous. In some embodiments, the sixth segment is heterologous.

In some embodiments, the present disclosure is directed to an RNAexpression cassette. In some embodiments, the expression cassettecomprises a target sequence, a first segment encoding a self-cleavingribozyme, and a second segment encoding a self-cleaving ribozyme. Insome embodiments, the target segment is flanked by the first segment andthe second segment. In some embodiments, the first self-cleavingribozyme is a 5′ self-cleaving ribozyme. In other embodiments, the firstself-cleaving ribozyme is a 3′ self-cleaving ribozyme. In someembodiments, the second self-cleaving ribozyme is a 5′ self-cleavingribozyme. In other embodiments, the second self-cleaving ribozyme is a3′ self-cleaving ribozyme. In some embodiments, the target segment isflanked by a 5′ self-cleaving ribozyme and a 3′ self-cleaving ribozyme.In some embodiments, the 5′ self-cleaving ribozyme and the 3′self-cleaving ribozyme are hammerhead ribozymes. In some embodiments,the 5′ self-cleaving ribozyme is a hammerhead ribozyme and the 3′self-cleaving ribozyme is a hepatitis delta virus (HDV) ribozyme. Insome embodiments, the target sequence comprises guide RNA (gRNA). Insome embodiments, the target sequence further comprises trans-activatingcrRNA (tracrRNA).

In some embodiments, the present disclosure is directed to a viralparticle. In some embodiments, the viral particle comprises a nucleicacid according to any aspect of the present disclosure. In someembodiments, the viral particle is a single-stranded RNA (ssRNA) virus.In some embodiments, the genome of the ssRNA virus is of negativepolarity. In some embodiments, the ssRNA virus is within the ordermononegavirales. In some embodiments, the ssRNA virus is a Sendai virus.In some embodiments, the ssRNA virus is attenuated. In some embodiments,the first region is inserted between intergenic elements. In someembodiments, the intergenic elements are P and M elements. In someembodiments, the second region is inserted between intergenic elements.In some embodiments, the intergenic elements are N and P elements. Insome embodiments, the viral particle comprises an RNA expressioncassette according to any aspect of the present disclosure. In someembodiments, the RNA expression cassette is located in the 3′ region ofthe viral genome. In some embodiemnts, the viral particle comprises atemperature sensitive mutant. In some embodiments, the viral particlecomprises a PL mutant. In some embodiments, the PL mutant does notstimulate host interferon production.

In some embodiments, the present disclosure is directed to a method ofintroducing target RNA into a host cell. In some embodiments, the hostcell is a prokaryotic cell. In some embodiments, the prokaryotic cellcomprises a bacterial or archaebacterial cell. In some embodiments, thehost cell is a eukaryotic cell. In some embodiments, the eukaryotic cellcomprises a plant cell, an animal cell, a protist, or a fungal cell. Insome embodiments, the animal cell comprises a vertebrate (chordate)cell. In some embodiments, the animal cell comprises an invertebratecell. In some embodiments, the animal cell comprises a mammalian cell.In some embodiments, the method comprises the steps of (i) contactingthe host cell with a viral particle according to any aspect of thepresent disclosure; and (ii) culturing the host cell under conditionsallowing (a) producing a target RNA; and (b) liberating the target RNA,wherein the first self-cleaving ribozyme liberates the target RNA fromthe transcribed first region. In some embodiments, the host cell isselected from the group consisting of an archaea cell, bacterial cell,and a eukaryotic cell.

In some embodiments, the present disclosure is directed to a method ofintroducing a site-specific modification to target DNA in a host cell.In some embodiments, the host cell is a prokaryotic cell. In someembodiments, the prokaryotic cell comprises a bacterial orarchaebacterial cell. In some embodiments, the host cell is a eukaryoticcell. In some embodiments, the eukaryotic cell comprises a plant cell,an animal cell, a protist, or a fungal cell. In some embodiments, theanimal cell comprises a vertebrate (chordate) cell. In some embodiments,the animal cell comprises an invertebrate cell. In some embodiments, theanimal cell comprises a mammalian cell. In some embodiments, the methodcomprises the steps of (i) contacting the host cell with a viralparticle according to any aspect of this disclosure where the viralparticle has a genome encoding a 5′ self-cleaving ribozyme, gRNA, and anuclease; (ii) culturing the host cell under conditions allowing (a)producing the gRNA flanked by the 5′ self-cleaving ribozyme and thenuclease; (b) liberating the gRNA, wherein the 5′ self-cleaving ribozymeliberates the gRNA, (c) expressing the nuclease; (d) forming a complexbetween the nuclease and the gRNA, wherein the scaffold sequence of thegRNA is bound to the nuclease; and (e) contacting the target DNA withthe complex, wherein the targeting sequence of the gRNA binds to asequence on the target DNA adjacent to a protospacer adjacent motif(PAM); and (iii) introducing the site-specific modification to thetarget DNA. In some embodiments, the site-specific modification is aninsertion. In some embodiments, the site-specific modification is adeletion. In some embodiments, the site-specific modification is aframeshift. In some embodiments, the site-specific modification is apoint mutation. In some embodiments, the site-specific modification isone of an insertion, a deletion, a frameshift, and a point mutation. Insome embodiments, the DNA is genomic. In some embodiments, the DNA ischromosomal. In other embodiments, the DNA is extra-chromosomal. In someembodiments, the DNA is mitochondrial DNA. In some embodiments, the DNAis chloroplast DNA. In some embodiments, the DNA is on a plasmid.

In some embodiments, the present disclosure is directed to a vector orvector system. In some embodiments, the vector comprises DNA encodingany nucleic acid of the present disclosure. In some embodiments, thevector is one or more plasmids. In some embodiments, the vector orvector system is one or more cosmids. In some embodiments, at least oneplasmid has a T7-driven promoter element. In some embodiments, thepresent disclosure is directed to a cell transformed with a vector orvector system of any aspect of the present disclosure. In someembodiments, the cell is a bacterial cell. In some embodiments, the cellis E. coli. In some embodiments, the cell is S. pyogenes. In someembodiments, the cell is a fungal cell. In some embodiments, the cell isS. cerevisiae or S. pombe. In some embodiments, the cell is P. pastoris.In some embodiments, expression of one or more vectors is concomitant.In other embodiments, expression of one or more vectors is separatelyinducible.

In some embodiments, the present disclosure is directed to a kit. Insome embodiments, the kit comprises a vector according to any aspect ofthe present disclosure. In some embodiments, the kit comprises apharmaceutically acceptable preservative or carrier. In someembodiments, the kit further comprises reagents for expressing the DNAencoding the genome sequence or the antigenome sequence that iscomplementary to the genome sequence. In some embodiments, the reagentsinclude polymerase. In some embodiments, the polymerase is T7 RNApolymerase. In some embodiments, the reagents include primers. In someembodiments, the kit further comprises instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents Sendai virus incorporating Cas9 and a guide RNA (gRNA)flanked by self-cleaving ribozymes replicates to high titer. FIG. 1A:The negative-sense RNA genome is flanked by virus promoters (the 3′leader (le), which serves as the genomic promoter, and the 5′ trailer(tr), which serves as the antigenomic promoter). Shown are the Sendaivirus genes N (nucleoprotein), P (phosphoprotein), M (matrix), F (fusionprotein), HN (attachment protein), and L (large RNA-dependent RNApolymerase). An EGFP-P2A-Cas9 cassette (5.1 kb) was inserted between Nand P, and a guide RNA flanked by self-cleaving ribozymes (rbz 1 and 2)(0.2 kb total) was inserted between P and M. The ribozymes are onlyfunctional in the positive-sense, or 5′-to-3′, orientation. Genome maybe transcribed from 3′ to 5′ into either full length antigenome orindividual capped and polyadenylated mRNAs. These mRNAs are produced ina polar transcriptional gradient, with N mRNAs being the most abundant,and L mRNAs being the least abundant. FIG. 1B: The self-cleavinghammerhead ribozyme sequences (SEQ ID NO:2 and SEQ ID NO:3) andstructures are shown. The chimeric guide RNA is shown in orange,corresponding to the orange highlight in FIG. 1A. Arrows indicate sitesof cleavage. FIG. 1C: The self-cleavage activity of the ribozymes wasassayed by qRT-PCR as described in Materials and Methods. Error barsrepresent standard deviation from 3 independent experiments. FIG. 1D:rSeV-Cas9 (WT), or rSeV-Cas9 with both ribozymes mutated to abolishself-cleavage (Mut), was rescued from plasmid DNA. As EGFP is onlyexpressed upon conversion of transfected antigenome to genome andsubsequent virus mRNA production, rescue efficiency was determined byobserving GFP+ cells (rescue events) by flow cytometry at 1-2 dayspost-transfection (dpt). Error bars represent standard deviation from 3replicates. ns, not significant. FIG. 1E: BSR-T7 cells were infected ata multiplicity of infection (MOI) of 0.01. Although the ribozymes inrSeV-Cas9 (WT) appear to affect growth compared to the mutant with noself-cleavage (Mut), they both reach the same peak titer of almost 10⁸IU/mL. FIG. 1F: HEK293 cells in 6-well were transfected with 2 ug px330(from which the FLAG-tagged Cas9 in rSeV-Cas9 was derived) or infectedwith rSeV-Cas9 at a MOI of 10. Cell lysates were collected 2 days laterand processed via SDS-PAGE and Western blot analysis for detection ofthe FLAG epitope on Cas9. COX IV represents the loading control.

FIG. 2 represents rSeV-Cas9 targeting mCherry gene achieves almostcomplete mutagenesis of a reporter cell line. FIG. 2A: mCherry-inducibleHEK293 cells were infected with rSeV-Cas9-control (no guide RNA) orrSeV-Cas9-mCherry (guide RNA targeting mCherry) at MOI 25. Expression ofmCherry was induced with doxycycline (dox) after 4 days post-infection,and cells were collected for flow cytometry the following day. Percentknockout (KO) of mCherry fluorescence was determined as100*(1−(C/(C+D)/(A/(A+B)). Results from 3 independent experiments areshown. FIG. 2B: Cells treated as in panel FIG. 2A were imaged byfluorescence microscopy. The same exposure was used for each condition.FIG. 2C: rSeV-Cas9-mCherry was mutated to render Rbz 2 (Rbz 2-mut) orboth ribozymes (Rbz 1/2-mut) non-functional. An alternative 3′ ribozyme,the hepatitis delta virus (HDV) ribozyme, was also tested viareplacement of Rbz 2. The experiment was performed as in FIG. 2A. FIG.2D: HEK293 cells were infected with rSeV-Cas9-control orrSeV-Cas9-mCherry at MOI 25 and collected for deep sequencing of themCherry locus at 6 days post-infection. Error bars represent Jeffreys95% confidence intervals. The 5 most abundant species of mutated target(SEQ ID NOs: 44-49, respectively) and their relative abundancepercentages are shown. Highlights represent the 20 bp target sequence,the arrowhead represents the Cas9 cleavage site, and the 3 bp PAM motifis shown.

FIG. 3 represents rSeV-Cas9 efficiently mutates endogenous ccr5 andefnb2. FIG. 3A: Affinofile cells were infected with rSeV-Cas9-control orrSeV-Cas9-CCR5 at MOI 25. CD4/CCR5 overexpression was induced at day 2,and cells were further infected with CCR5-tropic HIV-1 the followingday. Flow cytometry for p24 and CCR5 was performed 5 days afterinfection with rSeV. Data shown is gated on rSeV-infected cells (GFP+).FIG. 3B: HEK293 cells were infected with rSeV-Cas9-control or thetargeting viruses rSeV-Cas9-CCR5 or rSeV-Cas9-EFNB2 at MOI 25. Flowcytometry at 2 days post-infection indicated 98% infection. Cells werecollected at 6 days post-infection for deep sequencing of target andoff-target loci (see Table 2 infra for genomic locations and sequences).Error bars represent Jeffreys 95% confidence intervals. For each target,the 5 most abundant species of mutated target (SEQ ID NOs: 50-61,respectively) and their relative abundance percentages are shown.

FIG. 4 represents Ccr5-targeting rSeV-Cas9 edits primary human monocytesat high frequency. FIG. 4A: Primary human monocytes were infected withrSeV-Cas9-control or rSeV-Cas9-CCR5 at MOI 50 with simultaneousstimulation with GM-CSF and collected at 5 days post-infection for deepsequencing of on-target and off-target loci. Flow cytometry showed 98%infection. Error bars represent Jeffreys 95% confidence intervals. Foreach target, the 5 most abundant species of mutated target (SEQ ID NOs:62-67, respectively) and their relative abundance percentages are shown.FIG. 4B: Primary human monocytes from an independent donor were infectedas in panel a, and cells were collected at 5 days post-infection forflow cytometry of cell surface CCR5. Data shown is gated on infectedcells (GFP+).

FIG. 5 represents a time course of mCherry fluorescence knockout byrSeV-Cas9-mCherry. FIG. 5A: mCherry-inducible HEK293 cells were infectedwith rSeV-Cas9-control or rSeV-Cas9-mCherry at MOI 25. mCherryexpression was induced with doxycycline at the indicated dayspost-infection, and cells were collected for flow cytometry thefollowing day. FIG. 5B: Histograms of mCherry expression (gated oninfected GFP+ cells) are shown below as an alternative comparison.

FIG. 6 represents abundance of ccr5 mutation variants in monocytes (FIG.6A) and HEK293s (FIG. 6B). The relative abundance of all mutationvariants for ccr5 are shown in the pie charts. These specific variantsare also highlighted in the HEK293 pie chart. The distributions ofvariant abundance for the 100 most abundant variants for eithermonocytes or HEK293s are shown at right.

FIG. 7 represents a diagram of qRT-PCR primers used for ribozymecleavage assay.

FIG. 8 represents a schematic of the PL mutant generated in Example 2.FIG. 8A shows a schematic of the specific P and L mutations. FIG. 8Bshows the temperature sensitive phenotype of the PL mutants.

FIG. 9 represents the PL mutant's (ts rSeV-Cas9-CCR5 vector) ability toinfect human CD34+ hematopoietic stem cells (HSCs) from both human fetalliver and peripheral blood. FIG. 9A shows the same schematic from FIG.8A for reference. The gRNA is targeted against CCR5 and is the exactgRNA that was used in Example 1. FIG. 8B shows PL mutanttransduction/infection of purified human fetal liver CD34+ andperipheral blood mobilized CD34+ HSCs (>90% GFP+ at 2 dayspost-infection (dpi) using an MOI of 5; infection performed at 34° C.).FIG. 9C shows time course of infection at 34° C. vs 37° C. CD34+ HSCswere infected at 34° C. for 2 days and then either maintained at 34° C.or shifted to 37° C. at 2 dpi. The GFP+ cells steadily declined. FIG. 9Dshows Sanger sequencing data from PL mutant-infected CD34+ HSCs at 2dpi. 19/24 clones (˜80%) showed indels at the targeted CCR5 locus. Thewild type and first four clones have SEQ ID NOs: 68-72, respectively.

FIG. 10 represents the PL mutant (ts rSeV-Cas9-CCR5 vector) efficientlytransduces human CD34+/CD38−/CD45RA−/CD90+(Thy1+)/CD49fhigh cells(LT-HSC, SCID-Repopulating Cells). Phenotyping of infected CD34+ HSCsshowed that the PL mutants can infect >90% ofCD34+/CD38−/CD45RA−/CD90+(Thy1+)/CD49f-high cells which are known in theliterature as long-term-HSC or SCID-repopulating cells, capable ofreconstituting SCID (immuodeficient) mice at a single cell level (i.e.“true” stem cells.)

FIG. 11 represents the fold induction of 2 representative ISGs(interferon stimulated genes) in 293T cells infected with either the“wild type” rSeV-Cas9 vector or the PL mustants across a wide range ofviral inoculum. IFIT1 fold induction is represented in FIG. 11A. RIG-1fold induction is represented in FIG. 11B. Viral replication and ISGinduction was measured by qRT-PCR. Eveb at high viral genome copies, thePL mutant virus was markedly deficient in inducing ISGs. This remainedtrue regardless of the gRNA contained (mCherry or CCR5). Data is shownfor the CCR5 gRNA virus.

FIG. 12 represents a schematic diagram of an rSeV vector that candeliver two gRNAs, e.g. CCR5 gRNA and HRPT gRNA. FIG. 12A represents aschematic of the PL mutant. FIG. 12B represents a schematic of a PLmutant modified to be missing the Fusion protein (ΔF) and a target RNAdelivery region modified to deliver two gRNAs; CCR5 and HRPT, bothflanked by hammerhead and HDV self-cleaving ribozymes.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present disclosure relates to novel nucleic acids,including but not limited to, RNA expression cassettes. The nucleicacids generally relate to genetically modified genomes or antigenomescomplementary to the genomes of ssRNA viruses, e.g. the Sendai virus(SeV). The genome of mononegavirales, including but not limited toparamyxoviruses and SeV, is antisense RNA (i.e. having negativepolarity). Thus, when the present specification refers to a nucleic acidcomprising a genome sequence of a single-stranded RNA (ssRNA) virus, thegenome sequence is understood as being antisense RNA. The antigenomesequence is therefore sense RNA. Accordingly, an RNA antigenometranscribed from the negative polarity genome of, e.g. Sendai virus,will contain sense RNA. mRNA transcribed from the genome of an ssRNAvirus will likewise contain sense RNA that can be translated, or in theinstance where the mRNA contains a target sequence adjacent to one ormore self-cleaving ribozymes (e.g. flanked by self-cleaving ribozymes),the self-cleaving ribozymes will be able to liberate the targetsequence. Because the antigenome of the present disclosure is orientedin the 5′ to 3′ direction, similar to the biologically active mRNAtranscribed from the negative polarity genome, elements of the nucleicacids of the present disclosure are typically referred to by theirantigenome components. However, the invention as described herein isexplicitly not limited to just the antigenome components, as the genomiccomponents, e.g. as contained within the viral particles of the presentdisclosure (discussed infra) represent novel nucleic acids that may betranscribed in vitro or in vivo into an antigenome or antigenomicelements thereof or transcribed into mRNA fragments which undertake abiologically active role.

The antigenome sequences of the present disclosure generally comprise afirst region comprising (i) a target segment, and (ii) a first segmentencoding a first self-cleaving ribozyme. The target segment is to beunderstood as being a payload, e.g. an RNA payload, that is capable ofbeing liberated from mRNA transcribed from the genome by the one or moreself-cleaving ribozymes adjacent to the target segment. Thisself-cleaving ribozyme may be either a 5′ self-cleaving ribozyme or a 3′self-cleaving ribozyme. Preferred embodiments of the present disclosureutilize both a 5′ self-cleaving ribozyme and a 3′ self-cleaving ribozymethat flank the target segment to be delivered to the cell. FIG. 2Cillustrates that while it is preferable to have both 5′ and 3′self-cleaving ribozymes present, only one is truly necessary for theinvention to work in this aspect. However, while not wishing to be boundby theory, it is believed that for methods involving introduction ofsite-specific modifications to target DNA (i.e. CRISPR-relatedapplications), at least one 5′ self-cleaving ribozyme must be present.This is because the targeting sequence of gRNA is located on the 5′ endof the gRNA and small alterations to such may result in a loss oftargeting ability. However, for general delivery of target segments,e.g. target RNA to a cell, either a single 5′ self-cleaving ribozyme ora 3′ self-cleaving ribozyme will be sufficient. The general concept ofthe present disclosure with respect to this aspect is that the genomeand the antigenome sequences can “store” (i.e. encode) one or moreself-cleaving ribozymes without being active, as they are onlybiologically active once transcribed into mRNA from the genomesequences. While not wishing to be bound by theory, it is believed thatalthough the antigenome contains the ribozyme(s) in the same orientationas mRNA transcribed from the genome, the self-cleaving ribozymes are notactive in the antigenome. This is believed to be due toco-transcriptional encapsidation of the genomic and antigenomic RNA bythe viral nucleocapsid protein. This is significant because if theantigenome was self-cleaving, production of additional full-lengthgenomes from the antigenome would be impossible. Therefore, theribozymes of the present disclosure are considered only active in themRNAs transcribed from the genome, which are not encapisdated by thenucleocapsid protein which encapisdates the viral antigenome(represented by N in FIG. 1). Once mRNA is transcribed from the viralgenome, the self-cleaving ribozymes activate and cleave themselves andby doing so, liberate the target segment that they are either adjacentto (in the case of a single self-cleaving ribozyme) or flank (in thepreferred case of two self-cleaving ribozymes). The target segment isthen free to serve any particular utility in the cell which transcribedthe viral genome.

The self-cleaving ribozymes of the present disclosure can take a numberof forms. The exemplary self-cleaving ribozymes are hammerheadribozymes, as shown in FIG. 1A. However, other self-cleaving ribozymesmay be used, including hepatitis delta virus (HDV) ribozymes. It isimportant to note that as detailed herein, HDV ribozymes may only beutilized as 3′ self-cleaving ribozymes, whereas hammerhead ribozymes maybe utilized in both 5′ and 3′ self-cleaving ribozymes. Otherself-cleaving ribozymes which may be utilized include twister (e.g.Twiser from O. sativa, env9, and env22), twister-sister, pistol,hatchet, hairpin, Neurospora VS, and glmS ribozymes. Self-cleavingribozymes are generally characterized by distinct active sitearchitectures and divergent, but similar, biochemical properties. Thecleavage activities of self-cleaving ribozymes are highly dependent upondivalent cations, pH, and base-specific mutations, which can causechanges in the nucleotide arrangement and/or electrostatic potentialaround the cleavage site. Self-cleaving ribozymes are detailed inWeinberg et al., 2015, Nature Chemical Biology, “New classes ofself-cleaving ribozymes revealed by comparative genomics analysis” andLee et al., 2017, Molecules, “Structural and Biochemical Properties ofNovel Self-Cleaving Ribozymes,” both references hereby incorporated byreference in their entireties. Without wishing to be bound by theory,the mechanism of action of most self-cleaving ribozymes is based inacid-base catalysis of guanine and adenine in close proximity of thecleavage site. Additionally, metal ions are believed to play astructural rather than catalytic role, despite the fact that somecrystal structures have shown a direct metal ion coordination to anon-bridging phosphate oxygen at the cleavage site. As new self-cleavingribozymes arise, they too will be considered to be within the scope ofthis disclosure, so long as they are capable of self-cleaving andsuccessfully delivering target segments (e.g. RNA payloads) to a targetcell when transcribed from a ssRNA viral genome.

As an exemplary method of use of the novel nucleic acids, the targetsegments may comprise guide RNA (gRNA), and/or tracrRNA. In suchembodiments, the nucleic acids may further (but not necessarily)comprise a second region. The second region of the genome may contain anucleotide sequence that, when transcribed, produces mRNA that iscapable of being translated to code for a nuclease, e.g. Cas9, includingbut not limited to Cas9 homologs, or Cpf1, although other nucleases maybe suitable for incorporation into the present disclosure. An exemplaryembodiment of this nucleic acid is shown at FIG. 1A. In suchembodiments, the first region and the second region may be subclonedinto different locations within the ssRNA viral genome. One particularconsideration in where to subclone the first region/second region is therate of transcriptional activity relative to genome location. Forexample, with SeV, an exemplary ssRNA virus, transcription is moreactive in the 3′ region of the viral genome, thus a region (orexpression cassette) that is located in the 3′ region of the genome willbe overexpressed relative to an region (or expression cassette) locatedwithin the 5′ region of the viral genome. This trait is common to allparamyxoviruses, which carries with it the strong implication thatsuccess achieved with SeV, as shown in Example 1 infra, translates tothe other members of the family. Thus there may be various benefits tocloning such regions closer to or farther from the 5′ or 3′ end of theviral genome. The takeaway is that the location of cloning into theviral genome of the first region and the second region is discretionary,and that the emphasis should be on the composition of the regionsthemselves, and not the remainder of the viral genome elements.

Another aspect of the present disclosure relates to viral particles thatcomprise the foregoing nucleic acids discussed in the above sectiontitled “nucleic acids.” As mentioned supra, the nucleic acids of thepresent disclosure relate to modified genomes and antigenomes of ssRNAviruses. Thus, this aspect of the disclosure relates to the modifiedssRNA viruses. As previously discussed, any ssRNA virus where the RNA isnegative polarity (i.e. antisense) is considered to be within the scopeof this disclosure and thus suitable for use. Explicitly, the viruses ofthe order mononegavirales are considered to be within the scope of thisdisclosure. This is because viruses of the order mononegavirales havecommon attributes that lend them suitable to incorporate the nucleicacids of the present disclosure. Mononegavirales possess a linear,single-stranded, non-infectious RNA strand having negative polarity.Mononegavirales have characteristic gene order, produce 5-10 distinctmRNAs via polar sequential transcription, and replicate by synthesizingcomplete antigenomes.

Families within mononegavirales include bornaviridae, filoviridae,nyamiviridae, paramyxodiridae, and rhabdoviridae. Genera withinbornaviridae include bornavirus. Genera within filoviridae includecuevavirus, ebolavirus, and marburgvirus. Genera within nyamiviridaeinclude nyavirus. Genera within paramyxoviridae includeaquaparamyxovirus, avulavirus, feravirus, heniparvirus, morbillivirus,respirovirus, rubulavirus, pneumovirus, and metapneumovirus. Generawithin rhabdoviridae include alemndravirus, baiavirus, curiovirus,cytorhabdovirus, dichorhavirus, ephemerovirus, hapavirus, ledantevirus,lyssavirus, novirhabdovirus, nuclearhabdovirus, perhabdovirus,sawgravirus, sigmavirus, sprivivirus, tibrovirus, tupavirus, andvesiculovirus. While one of ordinary skill in the art will readilyrealize that not all of these candidates may be as suitable forincorporation into certain embodiments of the present disclosure asparamyxoviridae, including but not limited to Newcastle disease virus(NDV) and the Sendai virus, each of these viruses possess the necessaryfeatures from a compositional standpoint to incorporate the nucleicacids of the present disclosure.

Viral particles of the present disclosure may be generated by routinesknown to those of ordinary skill in the art. For example, the viralparticles of the present disclosure may be generated from packagingcells that are transfected with vectors, e.g. one or more plasmids ofthe present disclosure, that contain DNA encoding a nucleic acid of thepresent disclosure, e.g. a viral genome or antigenome of the presentdisclosure. This allows for scalable production of the viral particles,especially in instances where the viral particles are attenuated, e.g.not replication-competent. Expression of the vectors is induced in thepackaging cells and the viruses assemble for harvesting. This process isexemplified in Example 1, where E. coli cells were transfected withplasmid containing elements of the rSev-Cas9 recombinantgenome/antigenome. Other packaging cells are known to one of ordinaryskill in the art and may include, but are explicitly not limited to,HEK293 cells and PA317 cells.

Of the ssRNA viruses suitable for the present disclosure, the Sendaivirus (SeV) is of particular interest, and is an exemplary virus usedthroughout Example 1 infra. The present disclosure has surprisinglyshown that certain ssRNA viruses, e.g. paramyxoviruses, exemplified bythe Sendai virus, can tolerate self-cleaving ribozymes within thegenome. While not wishing to be bound by theory, as discussed supra,this is likely due to co-transcriptional encapsidation of the genomicand antigenomic RNA by the nucleoprotein and thus prevention of ribozymeactivity during replication of the full-length RNA. Along with furtherincorporation of Cas9 expression, the rescued replication-competentvirus was able to efficiently induce mutagenesis of the guide RNA targetsequence in the genome. For example, although the efficiency of theccr5-targeting virus is not directly comparable to other studies due tothe differing guide RNA sequences and target cells used, rates of ccr5mutagenesis (75-88%) was achieved similar to or higher than thoseachieved via lentivirus or AAV CRISPR/Cas9 transduction. Further,because infection with Sendai virus was highly efficient, achievingthese high rates of mutagenesis did not require sorting or selection forinfected cells, another distinct advantage.

In addition to the advantages of broad tropism, growth to high titers,and robust expression of foreign genes previously mentioned, ssRNAviruses, e.g. Sendai virus, have additional important advantages as agene therapy vector. First, such viruses are amenable to envelopeswitching or modification, in which envelope proteins with differentcell type specificities can be substituted for the original, or theoriginal attachment or fusion protein itself can be modified to have adifferent specificity. Second, Sendai virus, like other paramyxoviruses,has a polar transcriptional gradient (FIG. 1A) with reduction oftranscript levels as the polymerase complex moves from the 3′ to 5′ endof the genome. The efficiency versus the specificity of Cas9 activityappears to be a trade-off, and the optimal levels of Cas9 and guide RNAexpression therefore likely must be determined for each CRISPR deliveryplatform. Thus, for paramyxoviruses in particular, levels of Cas9 andguide RNA expression can be modulated and fine-tuned by shifting theinserted regions of these introduced elements within the genome, or bymodifying the strength of gene start signals. Third, paramyxoviruses arenot prone to genetic recombination or instability, and no homologous orheterologous recombination has ever been detected for Sendai virus.Fourth, despite a high prevalence of immunity to the related humanparainfluenza virus-1, cross-neutralizing anti-Sendai virus titers arelow. Thus, Sendai virus, as a mouse pathogen, would not encountersignificant pre-existing specific immunity in humans, making Sendaivirus in particular a highly attractive target for gene therapy, e.g.delivery of a RNA target to a cell.

Although the disclosure is strictly not limited to Sendai virus (SeV),the Sendai virus has several characteristics that render it surprisinglyeffective. The Sendai virus has been extensively studied and modified todevelop temperature-sensitive, non-cytopathic, andreplication-incompetent Sendai viruses that are useful for ex vivo andin vivo gene therapy applications. Mutations and variants of Sendaivirus have been characterized that allow replication of Sendai virus ata permissive temperature until a temporary shift to a non-permissivetemperature, after which replication is blocked and can no longer bedetected. Such control of Sendai viral replication with temperaturesensitivity can allow for temporal control of Cas9 and guide RNAexpression, which would reduce off-target effects by removing the vectoronce editing is complete, again speaking to the particular utility ofthe Sendai virus. Mutations that further confer the ability to avoidtriggering innate immune responses and concomitant cytopathogenicitywould avoid disturbing sensitive cell types such as hematopoietic stemcells or other primary cells. Finally, the Sendai virus is amenable tosingle and multiple deletions of the envelope and/or matrix genes suchthat the virus can only replicate when these viral factors are suppliedin trans. Upon infection of target cells in the absence of theseexogenously supplied factors, the virus can produce the factors encodedon its genome but cannot amplify via production of subsequent infectiousvirus.

Example 2 infra illustrates preferred embodiments of the recombinantSendai viral vectors, having mutant P and L genes, designated as PLmutants. The PL mutants are temperature sensitive, efficientlytransfecting at 34° C. but not at 37° C. More surprisingly, however, isthat the PL mutants do not induce a host interferon (IFN) response, i.e.do not stimulate production of IFN in a host when infected with the PLmutant vectors. The IFN-silent phenotype is particularly important whenapplying the viral vectors to sensitive cells like CD34+ hematopoieticstem cells where induction of IFN can drive differentiation andcompromise “sternness”. Accordingly PL mutants, particularly PL mutantsof the Sendai virus, represent a surprisingly effective vehicle for RNAtransfection in a host cell, e.g. stem cell.

The viral particles of the present disclosure may be utilized tointroduce an RNA payload into a target cell, e.g. a gRNA payload in thecase of CRISPR-related applications. Generally, the a host cell isinfected with a viral particle of the present disclosure and the hostcell is cultured under conditions that allow for the liberation of thetarget RNA. Cell culturing techniques are known to one of ordinary skillin the art. As detailed supra, transcription of the viral genome or aportion of the viral genome, e.g. transcription of an RNA expressioncassette inserted into the viral genome, into mRNA allows for the one ormore self-cleaving ribozyme(s) to cleave themselves out of thetranscribed mRNA and the target RNA along with it. In such embodiments,there need be only one self-cleaving ribozyme present, e.g. 5′self-cleaving ribozyme or a 3′ self-cleaving ribozyme. The self-cleavingribozyme must be adjacent to or flank the target RNA payload so that itis capable of liberating the payload upon transcription of the viralgenome into mRNA.

The target RNA may be, e.g., microRNA (miRNA), gRNA, or any other RNA.The RNA payload does not have to have any particular therapeutic use,but one of ordinary skill in the art can envision many such uses. Forexample, the target RNA may be involved in RNA silencing. The RNA may beutilized to regulate gene expression, e.g. post-transcriptionally. Somenon-limiting examples of a target RNA include siRNA and miRNA, which mayor may not have specific therapeutic uses. siRNA may be utilized for RNAinterference (RNAi) to promote gene silencing. miRNAs are used forsimilar therapeutic end means, and may represent a particularly usefultherapeutic non-CRISPR related application of the present disclosure.miRNAs are currently being utilized to treat many distinct types ofdiseases, from autoimmune disease to neurodegenerative disorders tocancer. miRNAs are typically endogenous 17-24 base-long single-stranded,non-coding RNAs that regulate gene expression in a sequence-specificmanner in plants and animals. Endogenously, miRNAs are derived fromlonger RNA transcripts by Drosha and Dicer. The resultant miRNAs bind totheir target sequence, typically within the 3′ untranslated region (UTR)of mRNA, thus leading to repression of translation. The presentdisclosure provides an alternative delivery mechanism for miRNA bysimply cleaving the miRNA from the transcribed antigenome, e.g. byflanking self-cleaving ribozyme(s). One of ordinary skill in the artwill appreciate that the class of miRNAs which can be delivered in thismanner are vast, and are not considered to be limited according to thetherapeutic use of such, rather they are to be considered within thescope of delivering a target RNA to a cell according to the presentdisclosure.

An exemplary use of the nucleic acids/viral particles of the presentdisclosure relates to gene editing via CRISPR-related technology. CRISPRstands for clustered regularly interspaced short palindromic repeatstype II system. CRISPR is a bacterial immune system that is modified forgenetic engineering purposes. Prior to CRISPR the most common genomicengineering approaches utilized zinc finger nucleases. CRISPR relies ontwo components, a guide RNA (gRNA) and a non-specific CRISPR-associatedendonuclease, e.g. Cas9. The gRNA is a synthetic RNA having a scaffoldsequence and a target sequence. The scaffold sequence is necessary forbinding to the nuclease, e.g., Cas9. The targeting sequence, oftenapproximately (although explicitly not necessarily) 20 nucleotides inlength, defines the genomic target to be modified. Thus, one of ordinaryskill in the art can change the genomic target by simply changing thetargeting sequence present in the gRNA. The genomic target can be any˜20 nucleotide DNA sequence, provided it meets two conditions: 1) thesequence is unique compared to the rest of the organism's genome and 2)the target is present immediately upstream of a Protospacer AdjacentMotif (PAM). The PAM sequence is dependent upon the exact species fromwhich the nuclease was originally derived from. For example, the PAM forCas9 derived from S. pyogenes is 5′-XGG-3′, wherein X is any nucleobase,whereas the PAM for Cfp1 is 5′-TTX-3′. One of ordinary skill in the artwill be familiar with different nucleases, e.g. Cas9 and relatedproteins, and their corresponding PAMs.

Cas9 was originally isolated from S. pyogenes, and while that remains anexemplary nuclease in the disclosure, there are many differentnucleases, including Cas9 variants, which are suitable for use in thisaspect of the disclosure. For example, there are synthetic Cas9 proteinsthat have artificial PAM recognition sequences, e.g. as described inKleinstiver B P et al., Nature, 2015 Jun. 23; 523(7561):481-5, herebyincorporated by reference in its entirety. There are Cas9 homologsderived from organisms other than S. pyogenes, for example, Cas9 from S.aureus (SaCas9). SaCas9 is approximately 1 kilobase smaller in size thanCas9 from S. pyogenes, which may render it more suitable forincorporation into the viral particles of the present disclosure due tothe limited genome size of some viral particles, although this is not anissue for Sendai virus as illustrated in Example 1 infra. One ofordinary skill in the art will appreciate that Cas9 derived from otherorganisms are only compatible with tracrRNA and crRNA or synthetic gRNAderived from the same host species. Furthermore, there are alternativesto Cas9 derived from S. pyogenes, synthetic Cas9, or Cas9 homologs. Onesuch alternative is Cpf1, described in Zetsche B et al., Cell. 2015 Oct.22; 163(3):759-71 and Kleinstiver B. P. et al., Nature Methods 2016 Aug.30; 714(13), both references incorporated by reference in theirentireties. Cpf1 has a PAM of 5′-TTX-3, wherein X is any nucleobase, andis located immediately upstream of the target DNA, instead of the targetDNA being immediately upstream of the PAM in the case of Cas9.Furthermore, Cpf1 cleavage results in a 5 nucleotide 5′ overhang 18 basepairs from the PAM sequence, whereas Cas9 cutting results in blunt DNAends 3 base pairs distal to the PAM sequence. Additionally, Cpf1 onlyrequires CRISPR RNA (crRNA) for successful targeting whereas Cas9requires both crRNA and transactivating crRNA (tracrRNA). Further CRISPRproteins may include C2c1, C2c2, and C2c3 proteins, disclosed in, forexample, Shmakov et al., Molecular Cell 2015 Oct. 22; 60(3): 385-397,hereby incorporated by reference in its entirety.

CRISPR-Cas gene editing systems have recently been reclassified into twoprimary classes spanning five types and sixteen subtypes, reviewed inMakarova, K., et al., Nature Reviews Microbiology 13:1-15 (2015), herebyincorporated by reference in its entirety. Classification was based uponidentifying all cas genes in a CRISPR-Cas locus and subsequentlydetermining key genes in each locus. This lead to a conclusion thatcurrently known CRISPR-Cas systems can classified as either “Class 1” or“Class 2” depending on the genes encoding the proteins involved in theinterference stage. A recent sixth CRISPR-Cas system has beenidentified, described in Abudayyeh O., et al. Science 2016, herebyincorporated by reference in its entirety.

“Class 1” systems generally comprise a multi-subunit crRNA-effectorcomplex, whereas “Class 2” systems generally comprise a single protein,such as Cas9, Cpf1, C2c1, C2c2, C2c3, or a crRNA-effector complex. Class1 systems comprise “Type I,” “Type III” and “Type IV” systems. “Class 2”systems comprise “Type II” and “Type V” systems. Class 1 CRISPR-Cassystems are characterized by effector modules consisting of multiplesubunits. Class 1 systems comprise about 90% of all CRISPR-Cas lociidentified in bacteria and archaea and can target both DNA and RNA, asdescribed in Makarova et al., Cell (2017) 168(5), hereby incorporated byreference in its entirety.

Type I systems are characterized by a Cas3 protein that has helicaseactivity and cleavage activity. Type I systems are further divided intoseven specific sub-types (I-A, I-B, I-C, I-D, I-E, I-F, and I-U). EachType I subtype has a defined combination of signature genes and distinctoperon organization. Type I systems additionally have a multiproteincrRNA-effector complex that is involved in the processing andinterference stages of the CRISPR-Cas immune system, known asCRISPR-associated complex for antiviral defense (“Cascade”). Sub-typeI-A comprises a csa5 gene which encodes a small subunit protein, a cas8gene that encodes degraded large and small subunits, and a split cas3gene. Archaeoglobus fulgidus is an exemplary organism with a sub-typeI-A CRISPR-Cas system. Sub-type I-B has a setcas1-cas2-cas3-cas4-cas5-cas6-cas7-cas8 gene arrangement while lacking acsa5 gene. Clostridium kluyveri is an exemplary organism with a sub-typeI-B CRISPR-Cas system. Sub-type I-C lacks a cash gene. Bacillushalodurans is an exemplary organism with a sub-type I-C CRISPR-Cassystem. Sub-type I-D has a cas10d gene instead of a cas8 gene.Cyanothece spp. is an exemplary organism with a sub-type I-D CRISPR-Cassystem. Sub-type I-E lacks a cas4 gene. Escherichia coli is an exemplaryorganism with a sub-type I-E CRISPR-Cas system. Sub-type I-F lacks acas4 gene and has a cas2 fused to a cas3. Yersinia pseudotuberculosis isan exemplary organism with a sub-type I-F CRISPR-Cas system. Geobactersulfurreducens is an exemplary organism with a sub-type I-U CRISPR-Cassystem.

All type III CRISPR-Cas systems have a cas10 gene, which encodes amultidomain protein containing a Palm domain, which is a variant of theRNA recognition motif (RRM), that is homologous to the core domain ofnumerous nucleic acid polymerases and cyclases and that is the largestsubunit of type III crRNA-effector complexes. Type III loci encode thesmall subunit protein, one Cas5 protein and typically several Cas7proteins. Type III are further divided into four sub-types, (III-A,III-B, III-C, and III-D). Sub-type III-A has a csm2 gene encoding asmall subunit and also has cas1, cas2 and cas6 genes. Staphylococcusepidermidis is an exemplary organism with a sub-type III-A CRISPR-Cassystem. Sub-type III-B has a cmr5 gene encoding a small subunit, lackingcas1, cas2 and cas6 genes. Pyrococcus furiosus is an exemplary organismwith a sub-type III-B CRISPR-Cas system. Sub-type III-C has a Cas10protein, but with an inactive cyclase-like domain, further lacking acast and cas2 gene. Methanothermobacter thermautotrophicus is anexemplary organism with a sub-type III-C CRISPR-Cas system. Sub-typeIII-D has a Cas10 protein that lacks the HD domain, further lacking acas1 and cas2 gene, but having a cas5-like gene known as csx10.Roseiflexus spp. is an exemplary organism with a sub-type III-DCRISPR-Cas system.

Type IV CRISPR-Cas systems encode a minimal multisubunit crRNA-effectorcomplex comprising a partially degraded large subunit, Csf1, Cas5, Cas7,and in some cases, a putative small subunit. Type IV systems lack castand cas2 genes. Type IV systems do not have sub-types, however there aretwo Type IV system variants. One Type IV variant has a DinG familyhelicase while the other does not, but the other has a gene encoding asmall α-helical protein. Acidithiobacillus ferrooxidans is an exemplaryorganism with a Type IV CRISPR-Cas system.

Type II CRISPR-Cas systems have cas1, cas2 and cas9 genes. The cas9 geneencodes the Cas9 protein, a multidomain protein that combines thefunctions of the crRNA-effector complex with target DNA cleavage. TypeII systems also encode a tracrRNA. Type II systems are further dividedinto three sub-types, sub-types II-A, II-B and II-C. Sub-type II-Acomprises the additional gene, csn2. Streptococcus thermophiles is anexemplary organism with a sub-type II-A CRISPR-Cas system. Sub-type II-Blacks the csn2 gene, but has the cas4 gene. Legionella pneumophilai isan exemplary organism with a sub-type II-B CRISPR-Cas system. Sub-typeII-C is the most common Type II system has only three proteins, Cas1,Cas2 and Cas9. Neisseria lactamica is an exemplary organism with asub-type II-C CRISPR-Cas system

Type V systems have a cpf1 gene and cas1 and cast genes. The cpf1 geneencodes a protein, Cpf1, that has a RuvC-like nuclease domain that ishomologous to the respective domain of Cas9, but lacks the HNH nucleasedomain that is present in Cas9 proteins. Type V systems have beenidentified in several bacteria, including Parcubacteria bacteriumGWC2011_GWC2_44_17 (PbCpf1), Lachnospiraceae bacterium MC2017 (Lb3Cpf1), Butyrivibrio proteoclasticus (BpCpf1), Peregrinibacteriabacterium GW2011 GWA 33_10 (PeCpf1), Acidaminococcus spp. BV3L6(AsCpf1), Porphyromonas macacae (PmCpf1), Lachnospiraceae bacteriumND2006 (LbCpf1), Porphyromonas crevioricanis (PcCpf1), Prevotelladisiens (PdCpf1), Moraxella bovoculi 237(MbCpf1), Smithella spp.SC_KO8D17 (SsCpf1), Leptospira inadai (L1Cpf1), Lachnospiraceaebacterium MA2020 (Lb2Cpf1), Franciscella novicida U112 (FnCpf1),Candidatus methanoplasma termitum (CMtCpf1), and Eubacterium eligens(EeCpf1). It has also been demonstrated that Cpf1 also has RNaseactivity and it is responsible for pre-crRNA processing, as disclosed inFonfara, I et al., Nature 28; 532(7600):517-21 (2016), herebyincorporated by reference in its entirety.

In Class 1 systems, the expression and interference stages involvemultisubunit CRISPR RNA (crRNA)-effector complexes. In contrast, inClass 2 systems, the expression and interference stages involve a singlelarge protein, e.g., Cas9, Cpf1, C2c1, C2c1, or C2c3, each of which isexplicitly considered within the scope of this invention.

In Class 1 systems, the expression and interference stages involvemultisubunit CRISPR RNA (crRNA)-effector complexes. In contrast, inClass 2 systems, the expression and interference stages involve a singlelarge protein, e.g., Cas9, Cpf1, C2c1, C2c1, or C2c3.

In Class 1 systems, pre-crRNA is bound to the multisubunitcrRNA-effector complex and processed into a mature crRNA. In Type I andIII systems this involves an RNA endonuclease, for example, Cash. InClass 2 Type II systems, pre-crRNA is bound to Cas9 and processed into amature crRNA in a step that involves RNase III and a tracrRNA. However,in at least one described Type II CRISPR-Cas system, crRNAs with mature5′-ends are directly transcribed from internal promoters where crRNAprocessing does not occur.

In Class 1 systems, the crRNA is associated with the crRNA-effectorcomplex and achieves interference by combining nuclease activity withRNA-binding domains and base pair formation between the crRNA and atarget nucleic acid.

In Type I systems, the crRNA and target binding of the crRNA-effectorcomplex involves Cas7, Cas5, and Cas8 fused to a small subunit protein.The target nucleic acid cleavage of Type I systems involves the HDnuclease domain, which is either fused to the superfamily 2 helicaseCas3′ or is encoded by a separate gene, cas3.

In Type III systems, the crRNA and target binding of the crRNA-effectorcomplex involves Cas7, Cas5, Cas10 and a small subunit protein. Thetarget nucleic acid cleavage of Type III systems involves the combinedaction of the Cas7 and Cas10 proteins, with a distinct HD nucleasedomain fused to Cas10, which, while not wishing to be bound by theory,is thought to cleave single-strand DNA during interference.

In Class 2 systems, the crRNA is associated with a single protein andachieves interference by combining nuclease activity with RNA-bindingdomains and base pair formation between the crRNA and a target nucleicacid.

In Type II systems, the crRNA and target binding involves Cas9 as doesthe target nucleic acid cleavage. In Type II systems, the RuvC-likenuclease (RNase H fold) domain and the HNH (McrA-like) nuclease domainof Cas9 each cleave one of the strands of the target nucleic acid. TheCas9 cleavage activity of Type II systems also requires hybridization ofcrRNA to tracrRNA to form a duplex that facilitates the crRNA and targetbinding by the Cas9.

In Type V systems, the crRNA and target binding involves Cpf1 as doesthe target nucleic acid cleavage. In Type V systems, the RuvC-likenuclease domain of Cpf1 cleaves one strand of the target nucleic acidand a putative nuclease domain cleaves the other strand of the targetnucleic acid in a staggered configuration, producing 5′ overhangs, whichis in contrast to the blunt ends generated by Cas9 cleavage. While notwishing to be bound by theory, these 5′ overhangs may facilitateinsertion of DNA through non-homologous end-joining methods.

As discussed herein, the Cpf1 cleavage activity of Type V systems alsodoes not require hybridization of crRNA to tracrRNA to form a duplex,rather the crRNA of Type V systems use a single crRNA that has a stemloop structure forming an internal duplex. Cpf1 binds the crRNA in asequence and structure specific manner, that recognizes the stem loopand sequences adjacent to the stem loop, most notably, the nucleotide 5′of the spacer sequences that hybridizes to the target nucleic acid. Thisstem loop structure is typically in the range of 15 to 19 nucleotides inlength. Substitutions that disrupt this stem loop duplex abolishcleavage activity, whereas other substitutions that do not disrupt thestem loop duplex do not abolish cleavage activity. In Type V systems,the crRNA forms a stem loop structure at the 5′ end and the sequence atthe 3′ end is complementary to a sequence in a target nucleic acid.

Other proteins associated with Type V crRNA and target binding andcleavage include Class 2 candidate 1 (C2c1) and Class 2 candidate 3(C2c3). C2c1 and C2c3 proteins are similar in length to Cas9 and Cpf1proteins, ranging from approximately 1,100 amino acids to approximately1,500 amino acids. C2c1 and C2c3 proteins also contain RuvC-likenuclease domains and have an architecture similar to Cpf1. C2c1 proteinsare similar to Cas9 proteins in requiring a crRNA and a tracrRNA fortarget binding and cleavage, but have an optimal cleavage temperature of50° C. C2c1 proteins target an AT-rich PAM, which similar to Cpf1, is 5′of the target sequence. In contrast, Class 2 candidate 2 (C2c2) does notshare sequence similarity to other CRISPR effector proteins, and wasrecently identified as a Type VI system. C2c2 proteins have two HEPNdomains and demonstrate ssRNA-cleavage activity. C2c2 proteins aresimilar to Cpf1 proteins in requiring a crRNA for target binding andcleavage, while not requiring tracrRNA. Also like Cpf1, the crRNA forC2c2 proteins forms a stable hairpin, or stem loop structure, that aidin association with the C2c2 protein.

Specifically regarding Class 2 Type II CRISPR Cas systems, a largenumber of Cas9 orthologs are known in the art as well as theirassociated polynucleotide components (tracrRNA and crRNA) (see, e.g.,Fonfara, I., et al., Nucleic Acids Research 42.4 (2014): 2577-2590, andChylinski K., et al., Nucleic Acids Research, 2014; 42(10):6091-6105,both references hereby incorporated by reference in their entireties.Cas9-like synthetic proteins are known in the art (see, e.g., U.S.2014/0315985 and U.S. 2016/0362667, both references hereby incorporatedby reference in their entireties). Aspects of the present disclosure canbe practiced by one of ordinary skill in the art following the guidanceof the specification to use Type II CRISPR Cas proteins and Cas-proteinencoding polynucleotides, including, but not limited to Cas9, Cas9-like,proteins encoded by Cas9 orthologs, Cas9-like synthetic proteins, andvariants and modifications thereof. Cognate RNA components of these Casproteins can be manipulated and modified for use in the practice of thepresent disclosure.

In CRISPR-Cas related embodiments, the target sequence comprises a gRNAsequence. The target sequence may also further comprise transactivatingcrRNA (tracrRNA), and may comprise other elements. Furthermore, in suchCRISPR-related embodiments, the viral genome and antigenome contains asecond region that includes a sequence encoding a nuclease, e.g. Cas9.The second region may or may not contain a sequence encoding a reportermolecule or any other additional sequences. As detailed supra, thesecond region, like the first region, may be subcloned practicallyanywhere in the viral genome. However, in the case of paramyxoviruses,e.g. the Sendai virus, one of ordinary skill in the art will take intoconsideration the relative rates of transcription. FIG. 1 represents anexemplary genome for this embodiment. In such embodiments, once theportion of the viral genome encoding the gRNA and one or more ribozymesis transcribed into mRNA containing the gRNA, the gRNA is liberated bythe ribozyme(s). At least a 5′ ribozyme must be present, but inpreferred embodiments (though not necessarily), a flanking 3′ ribozymeis present too. The type of the ribozymes utilized (hammerhead, HDV,etc.) can be according to any of the embodiments discussed herein. Oncethe gRNA is liberated, and after the mRNA sequence encoding the nucleaseis translated, the gRNA binds to the nuclease, e.g. Cas9, through thescaffold sequence. The nuclease undergoes a conformational change oncebound to the gRNA through the scaffold sequence which shifts thenuclease from an inactive conformation to an active DNA-bindingconformation. Importantly, the targeting sequence of the gRNA remainsexposed so that it may interact with the DNA binding site. The gRNA thendirects the bound complex to the target DNA sequence, immediatelyupstream of the PAM, to which the nuclease will cleave. Alterations tothe DNA sequence may then be introduced.

One of ordinary skill in the art will be generally familiar with how tointroduce an alternation to target DNA after a double-stranded break hasbeen introduced, however for exemplary purposes, the most commonpathways utilized are the non-homologous end joining (NHEJ) DNA repairpathway and the homology directed repair (HDR) pathway. These pathwaysallow for introduction of alterations, most commonly insertions ordeletions (“indels”) but these alterations may include deletions,additions, substitutions, frameshift mutations, or point insertions. Onepotential advantage of the NHEJ pathway over the HDR pathway is that,unlike HDR, the NHEJ pathway is active throughout the cell cycle and hasa higher capacity for repair, as there is no requirement for a repairtemplate. Furthermore, NHEJ also repairs most types of breaks withinminutes, which is significantly faster than HDR. However, HDR is themore accurate mechanisms of the two due to the requirement of highersequence homology between the damaged and intact donor strands of DNA.HDR can be error-free if the DNA template used for repair is identicalto the original DNA sequence at the location of the break. Thus, HDR canintroduce very specific mutations into the damaged DNA. The HDR pathwaygenerally follows the following steps. First, the 5′-ended DNA strand isresected at the break to create a 3′ overhang. This serves as asubstrate for proteins required for strand invasion and a further as aprimer for DNA repair synthesis. The invasive strand then displaces astrand of the homologous DNA duplex and pair with another. This resultsin the formation of hybrid DNA referred to as the displacement loop (Dloop). The recombination intermediates are then resolved to complete theDNA repair process. In contrast, the NHEJ pathway generally follows thefollowing steps. First, after a double-stranded break has beenintroduced, the broken ends are recognized by a heterodimer, e.g. aKu70/Ku80 heterodimer. The heterodimer will act as a scaffold forrecruitment of a kinase, e.g. DNA-PKcs and a ligase, as well as someaccessory factors, e.g. PAXX, XLF. This forms a paired end complex,which then ligates the compatible DNA ends together. NHEJ utilizes anumber of polymerases, e.g. Polμ and Polk, nucleases as well asstructure specific enzymes, e.g. Tdp2 and Aprataxin. The processing ofDNA ends is where mutations are introduced in the NHEJ pathway.

Another aspect of the present disclosure relates to vectors, aside fromthe viral particles of the present disclosure that comprise the nucleicacids of the present disclosure. The vectors may be DNA or RNA vectors.In an exemplary embodiment, the vectors comprise plasmids that containDNA encoding both the genome and the antigenome. The plasmids may beinduced to generate the viral particles. The vector may includeappropriate sequences for amplifying expression. In addition, theexpression vector preferably contains one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells such as dihydrofolate reductase or neomycin resistance foreukaryotic cell cultures, or such as tetracycline or ampicillinresistance in E. coli.

Some embodiments of the present disclosure are directed to cellstransformed with the plasmids. Any of the procedures known in the artfor introducing foreign nucleotide sequences into host cells may beused. Examples include the use of calcium phosphate transfection,polybrene, protoplast fusion, electroporation, nucleofection, liposomes,microinjection, naked DNA, plasmid vectors, viral vectors, both episomaland integrative, and any of the other well-known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell.

Another aspect of the present disclosure relates to kits comprising thevectors of the present disclosure. The kits may further includereagents. In an exemplary embodiment, the reagents include T7 RNApolymerase. The kits may contain controls. The kits may containinstructions or directions for use. The kit may be comprised of one ormore containers and may also include collection equipment, for example,bottles, bags (such as intravenous fluids bags), vials, syringes, andtest tubes. Other components may include needles, diluents and buffers.Usefully, the kit may include at least one container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. Optionally, the kits of thedisclosure further include software to expedite the generation, analysisand/or storage of data, and to facilitate access to databases. Thesoftware includes logical instructions, instructions sets, or suitablecomputer programs that can be used in the collection, storage and/oranalysis of the data. Comparative and relational analysis of the data ispossible using the software provided.

The terms “conservative sequence modifications” or “conservativesubstitutions” as used herein may refer to nucleotide substitutions thatdo not significantly affect or alter the activity or characteristics ofthe self-cleaving ribozymes of the present disclosure.

The term “CRISPR” as used herein may refer to “clustered regularlyinterspaced short palindromic repeat”, which in the scope of the presentdisclosure is understood to be utilized in conjunction with a nucleasesuch as Cas9 to edit a target DNA sequence, e.g. CRISPR/Cas9 system. Theterm “Cas” as used herein may refer to “CRISPR associated protein”, andincludes but is not limited to the nuclease Cas9 and Cas9 proteins.“Cas9” or “Cas9 protein” as used herein includes Cas9 wild-type proteinderived from CRISPR-Cas9 systems, modifications of Cas9 proteins,analogs of Cas9 proteins, variants of Cas9 proteins, proteins expressedby cas9 orthologs, and combinations thereof. Other “Cas” proteins areknown in the art and are considered to be within the scope of thisdisclosure, including Cas9-like synthetic proteins, Cpf1 proteins(including wild-type Cpf1), Cpf1-like synthetic proteins, C2c1 proteins,C2c2 proteins, C2c3 proteins, and variants and modifications thereof“Cpf1” or “Cpf1 proteins” as used herein includes Cpf1 wild-type proteinderived from CRISPR-Cpf1 systems, modifications of Cpf1 proteins,variants of Cpf1 proteins, Cpf1 analogs, proteins expressed by cpf1orthologs, and combinations thereof.

As used herein, the term “guide RNA” or “gRNA” may refer to an RNAmolecule that can bind to a nuclease and guide the nuclease to aspecific location within a target DNA. A guide RNA can comprise twosegments: a “targeting sequence” and a “scaffold sequence”. “Targetingsequence” as used herein may refer to a nucleotide sequence that iscomplementary to, or at least can hybridize to under stringentconditions, a target DNA sequence. The protein-binding segment binds tonuclease, e.g. Cas9, Cpf1, or a related CRISPR associated protein(“Cas”) disclosed herein. The targeting sequence and the scaffoldsequence can be located in the same RNA molecule or in two or moreseparate RNA molecules.

The term “heterologous” as used herein may refer to biological elementsthat are from different sources, e.g. foreign DNA or RNA introduced intoan organism. For example, in the present disclosure, a nuclease may befrom a first source, e.g. Cas9 from S. pyogenes bacterium. Or, a targetsequence, e.g., gRNA may be from a second organism or may be synthetic.One of ordinary skill in the art will appreciate that introduction offoreign genetic material into an organism, e.g. introduction of anexpression cassette, may introduce many heterologous elements to theorganism.

The term “homology” as used herein may refer to the existence of sharedstructure between two compositions. The term “homology” in the contextof proteins may refer to the amount (e.g. expressed in a percentage) ofoverlap between two or more amino acid and/or peptide sequences. In thecontext of nucleic acids, the term may refer to the amount (e.g.expressed in a percentage) of overlap between two or more nucleic acidsequences. As used herein, the percent (%) homology between twosequences is equivalent to the percent identity between the twosequences. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e., %homology=# of identical positions/total # of positions×100), taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm. Such homology iswell-represented in the art via local alignment tools and/or algorithms,and may include pairwise alignment, multiple sequence alignment methods,structural alignment methods, and/or phylogenetic analysis methods.

As used herein, the term “expression” refers to transcription of apolynucleotide from a DNA template, resulting in, for example, an mRNAor other RNA transcript (e.g., non-coding, such as structural orscaffolding RNAs). The term further refers to the process through whichtranscribed mRNA is translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be referred to collectively as“gene products.” Expression may include splicing the mRNA in aeukaryotic cell, if the polynucleotide is derived from genomic DNA.

The terms “polynucleotide”, “nucleotide sequence” or “nucleic acid” asused herein may refer to a polymer composed of a multiplicity ofnucleotide units (ribonucleotide or deoxyribonucleotide or relatedstructural variants) linked via phosphodiester bonds, including but notlimited to, DNA or RNA. The term encompasses sequences that include anyof the known base analogs of DNA and RNA. Examples of a nucleic acidinclude and are not limited to mRNA, miRNA, tRNA, rRNA, snRNA, siRNA,dsRNA, cDNA and DNA/RNA hybrids. Nucleic acids may be single stranded ordouble stranded, or may contain portions of both double stranded andsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA, or a hybrid, where the nucleic acid may contain combinationsof deoxyribo- and ribo-nucleotides, and combinations of bases includinguracil (U), adenine (A), thymine (T), cytosine (C), guanine (G), andtheir derivative compounds. Nucleic acids may be obtained by chemicalsynthesis methods or by recombinant methods. The depiction of a singlestrand also defines the sequence of the complementary strand. Thus, anucleic acid also encompasses the complementary strand of a depictedsingle strand. Many variants of a nucleic acid may be used for the samepurpose as a given nucleic acid. Thus, a nucleic acid also encompassessubstantially identical nucleic acids and complements thereof.

The term “ribozyme” as used herein refers to RNA molecules that arecapable of catalyzing specific biochemical reactions. The activity of aribozyme is similar to that of a protein enzyme, the chief differencebeing the composition of the two. The term “self-cleaving ribozyme” asused herein refers to a RNA molecule motif that catalyzes cleavage andrelated reactions at a specific site within an RNA polymer. Examples ofself-cleaving ribozymes include but are not limited to hammerheadribozymes, hepatitis delta virus (HDV) ribozymes, twister ribozymes,twister sister ribozymes, pistol ribozymes, hairpin irobzymes, andhatchet ribozymes.

The term “treating” or “treatment” of a disease refers to executing aprotocol, which may include administering one or more drugs to a patient(human or otherwise), in an effort to alleviate signs or symptoms of thedisease. Alleviation can occur prior to signs or symptoms of the diseaseappearing as well as after their appearance. Thus, “treating” or“treatment” includes “preventing” or “prevention” of disease. The terms“prevent” or “preventing” refer to prophylactic and/or preventativemeasures, wherein the object is to prevent or slow down the targetedpathologic condition or disorder. In addition, “treating” or “treatment”does not require complete alleviation of signs or symptoms, does notrequire a cure, and specifically includes protocols that have only amarginal effect on the patient.

As used herein, a “host cell” generally refers to a biological cell. Acell can be the basic structural, functional and/or biological unit of aliving organism. A cell can originate from any organism having one ormore cells. Examples of host cells include, but are not limited to: aprokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell,a cell of a single-cell eukaryotic organism, a protozoa cell, a cellfrom a plant, an algal cell, a fungal cell (e.g., a yeast cell), ananimal cell, a cell from an invertebrate animal, a cell from avertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cellfrom a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, amouse, a non-human primate, a human, etc.). A host cell can be a stemcell or progenitor cell.

The term “patient” as used herein may refer to a biological system towhich a treatment can be administered. A biological system can include,for example, an individual cell, a set of cells (e.g., a cell culture),an organ, a tissue, or a multi-cellular organism. A “patient” can referto a human patient or a non-human patient.

The term “protospacer adjacent motif” or “PAM” as used herein refers tothe DNA sequence immediately following the target DNA sequence that istargeted by the nuclease in a CRISPR application setting. The PAM isnuclease specific, and the nuclease will not successfully bind to orcleave the target DNA sequence if it is not followed by the PAM. Theterm “vector” as used herein may refer to a nucleic acid sequencecontaining an origin of replication. A vector may be a plasmid,bacteriophage, bacterial artificial chromosome, yeast artificialchromosome or a virus. A vector may be a DNA or RNA vector. A vector maybe either a self-replicating extrachromosomal vector or a vector whichintegrates into a host genome. The term “expression vector” refers to anucleic acid assembly containing a promoter which is capable ofdirecting the expression of a sequence or gene of interest in a cell.Vectors typically contain nucleic acid sequences encoding selectablemarkers for selection of cells that have been transfected by the vector.Generally, “vector construct,” “expression vector,” and “gene transfervector,” refer to any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells or host cells.

As used herein and in the appended claims, the singular forms “a”, “and”and “the” include plural references unless the context clearly dictatesotherwise.

The term “about” refers to a range of values which would not beconsidered by a person of ordinary skill in the art as substantiallydifferent from the baseline values. For example, the term “about” mayrefer to a value that is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, as well asvalues intervening such stated values.

Publications disclosed herein are provided solely for their disclosureprior to the filing date of the present disclosure.

Each of the applications and patents cited in this text, as well as eachdocument or reference, patent or non-patent literature, cited in each ofthe applications and patents (including during the prosecution of eachissued patent; “application cited documents”), and each of the PCT andforeign applications or patents corresponding to and/or claimingpriority from any of these applications and patents, and each of thedocuments cited or referenced in each of the application citeddocuments, are hereby expressly incorporated herein by reference intheir entirety. More generally, documents or references are cited inthis text, either in a Reference List before the claims; or in the textitself; and, each of these documents or references (“herein-citedreferences”), as well as each document or reference cited in each of theherein-cited references (including any manufacturer's specifications,instructions, etc.), is hereby expressly incorporated herein byreference.

The following non-limiting examples serve to further illustrate thepresent disclosure.

EXAMPLES Example 1: Sendai Virus Delivers CRISPR/Cas9 for Gene EditingA. Materials and Methods

Cell Lines

Flp-In T-REx HEK293 cells (Invitrogen), Vero cells (ATCC CCL-81), BSR-T7cells (BHK-based cell line with stable expression of T7 polymerase), andAffinofile cells (HEK293-based cell line with inducible overexpressionof CD4 and CCR5) were propagated in Dulbecco's modified Eagle's medium(Invitrogen) supplemented with 10% fetal bovine serum (FBS) (AtlantaBiologicals) and penicillin/streptomycin at 37° C. Flp-In T-REx HEK293cells were additionally maintained in blasticidin and zeocin accordingto manufacturer protocol, BSR-T7 cells were additionally maintained in 1mg/mL G418 to maintain the T7 transgene, and Affinofile cells wereadditionally maintained in 50 μg/mL blasticidin. To generate themCherry-inducible cells, the mCherry gene was inserted intopcDNA5/FRT/TO and co-transfected with pOG44 (Flp-recombinase) intoparental Flp-In T-REx HEK293 cells. Selection with hygromycin (replacingzeocin) and blasticidin according to manufacturer protocol yielded astable cell line with doxycycline-inducible expression of mCherry.

Whole human blood was obtained from the New York Blood Center.Peripheral blood mononuclear cells were isolated using Ficoll-Paque (GEHealthcare), and monocytes were further purified using CD14 MicroBeads(Miltenyi Biotec). Monocytes were propagated in RPMI 1640 medium(Invitrogen) supplemented with 10% FBS (Atlanta Biologicals).

Sendai Virus Reverse Genetics Plasmids

The basis for rSeV-Cas9 was a recombinant Sendai virus with an EGFPreporter inserted between the N and P genes via duplication of theN-to-P intergenic region, derived from RGV0, a Fushimi strain SeV withmutations in the F and M genes that allow trypsin-independent growth.All modifications to the plasmid encoding the T7-driven antigenome wereperformed using standard and overlapping PCRs with Velocity DNApolymerase (Bioline), with subsequent insertion into the construct atunique restriction sites by In-Fusion ligation-independent cloning(Clontech). All cloning was performed with Stb12 E. coli (Invitrogen)with growth at 30° C. FLAG-tagged codon-optimized S. pyogenes Cas9 wasamplified from px330 (Addgene, cat #42230) and inserted into rSeVfollowing the EGFP reporter, linked with a P2A ribosomal skippingsequence (ATNFSLLKQAGDVEENPGP) (SEQ ID NO: 4). The P2A sequence waspreceded by a GSG linker to ensure complete ribosomal skipping. Anadditional two nucleotides were added after the stop codon of Cas9 tomaintain the rule of six, by which the genome length of paramyxovirusesmust be an exact multiple of six to ensure efficient replication. TheCas9 is flanked by unique NotI and FseI restriction sites to aid in anyfuture modifications. To create the guide RNA and ribozyme cassette, themCherry-targeting 20 bp sequence was cloned into px330 (discussedsupra), and the full chimeric guide RNA cassette (SEQ ID NO: 1, seeTable 1 below) was then PCR-amplified. The hammerhead ribozymes wereincorporated via overhangs in the synthesized primers in subsequentPCRs. This cassette was inserted between the P and M genes viaduplication of the P-to-M intergenic region, with unique AsiSI and SnaBIrestriction sites flanking the cassette to aid in future modificationsincluding changing the guide RNA target sequence. The guide RNA targetsequences were chosen based on high predicted specificity using a CRISPRdesign tool available online through MIT.

(SEQ ID NO: 1, Guide RNA Cassette)ATCCCGGGTGAGGCATCCCACCATCCTCAGTCACAGAGAGACCCAATCTACCATCAGCATCAGCCAGTAAAGATTAAGAAAAACTTAGGGTGAAAGAAATTTCACCTAACACGGCGCAGCGATCGCGTGGCCCTGATGAGTCCGTGAGGACGAAACGGTAGGAATTCCTACCGTCGGCCACGAGTTCGAGATCGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACGTATCACCGGAGTCGACTCCGGTCTGATGAGTCCGTGAGGACGAAATACGTATCCCGGGTGAGGCATCCCACCATCCTCAGTCACAGAGAGACCCAATCTACCATCAGCATCAGCCAGTAAAGATTAAGAAAAACTTAGGGTGAAAGAAATTTCACCTAACACGGCG CA

TABLE 1 Annotated sequence of gRNA Cassette. Nucleotide Positions WithinGuide RNA Cassette (SEQ ID NO: 1) Description  1-118, 325-442 P-to-Mintergenic region; guide RNA cassette inserted via duplication of thisregion in this instance  73-83, 397-407 Gene stop signal  84-86, 408-410Intergenic trinucleotide  87-96, 411-420 Gene start signal 119-126Restriction site, e.g. modified AsiSI restriction site in this instance127-132, 176-181 Stem for 5′ ribozyme (e.g. rbz 1). Note: 1^(st) part ofstem, e.g. GTGGCC in this instance, must be the reverse complement ofthe beginning of the gRNA targeting sequence. See, e.g., FIG. 1B forstem structure. 133-175 5′ ribozyme (e.g. rbz 1) 176-195 Guide RNA(gRNA), e.g. mCherry-targeting sequence in this instance 196-271TracrRNA 272-277, 319-324 Stem for 3′ ribozyme (e.g. rbz 2) 278-318 3′ribozyme (e.g. rbz 2) 320-325 Restriction site, e.g. modified SnaBIrestriction site in this instance 135 Mutated to C to abolish 5′ribozyme activity 299 Mutated to C to abolish 3′ ribozyme activity

Cleavage Assay

qRT-PCR primers were designed to flank ribozyme 1 (product A), ribozyme2 (product B), and within the downstream M gene (product C, representingtotal RNA) (see FIG. 7). rSeV-Cas9-mCherry T7-driven antigenome plasmidwas transfected into T7-expressing BSR-T7 cells for 2 hours beforecollection in TRIzol (Invitrogen). Samples were treated with DNase(Invitrogen) at 1 mM MgCl₂, treated with EDTA, and reverse-transcribedat 1 mM MgCl₂ with the SuperScript III First-Strand Synthesis System(Invitrogen). qRT-PCR was performed with the SensiFAST SYBR &Fluorescein kit (Bioline), with copy numbers determined by standardcurves using the rSeV-Cas9-mCherry antigenome plasmid as template.Percent ribozyme 1 cleavage was determined as 100*((C-A)/C) andnormalized to the construct with both ribozymes mutated, and percentribozyme 2 cleavage was determined as 100*((C−B)/C) and normalized tothe construct with ribozyme 2 mutated.

Viruses and Infections

BSR-T7 cells in 6-well were transfected with 4 ug T7-driven antigenome,1.44 μg T7-N, 0.77 μg T7-P, 0.07 μg T7-L, and 4 μg codon-optimized T7polymerase, using Lipofectamine LTX (Invitrogen) according tomanufacturer's recommendations. Virus rescue was monitored by appearanceand spread of EGFP fluorescence, and rescued virus was further expandedon BSR-T7 cells. Stocks of clarified virus were stored at −80° C. Virustiters were determined by titration on Vero cells, with individualinfection events detected and counted by EGFP fluorescence at 24 hourspost-infection in an Acumen plate reader (TTP Labtech).

For SeV infection of HEK293-based cell lines, 5×10⁴ cells were mixedwith the virus inoculum immediately prior to plating inpoly-L-lysine-coated wells. Media was changed the following day andevery 2 days thereafter. For induction of mCherry, 100 ng/mL doxycyclinewas used. For Affinofile cells, 2 μg/mL ponasterone A and 8 ng/mLdoxycycline were used to induce CCR5 and CD4, respectively. For furtherHIV-1 infection of Affinofile cells, JR-FL HIV-1 was spinoculated ontocells at 2000 rpm for 2 hours at 37° C. in the presence of 2 μg/mLpolybrene (Sigma).

For SeV infection of monocytes, virus stocks were further purified byultracentrifugation into a discontinuous 20% to 65% sucrose gradient.The interface was collected, titered on Vero cells, and stored at −80°C. until use. 5×10⁵ cells in serum-free medium were plated for 30minutes at 37° C. to allow adherence before infection with virusinoculum via spinoculation at 2000 rpm for 2 hours at 37° C. Media waschanged to RPMI with 10% FBS following spinoculation and changed every 2days thereafter. 100 ng/mL GM-CSF (Peprotech) was included in the mediafollowing infection to stimulate macrophage differentiation andconcomitant upregulation of CCR5.

Flow Cytometry

For CCR5 staining, cells were lifted and blocked in phosphate-bufferedsaline (PBS) with 2% FBS. Alexa 647-conjugated rat anti-human CCR5(BioLegend, cat #313712) was added at 1:100 for 30 minutes at 4° C.before washing and resuspension in 2% paraformaldehyde (PFA). For p24staining (RD1-conjugated mouse anti-p24 clone KC57, Beckman Coulter, cat#6604667, 1:100 dilution), cells were fixed and permeabilized using theCytofix/Cytoperm kit (BD Biosciences) before blocking. Flow cytometrywas performed on a BD LSR II at the Flow Cytometry Core at the IcahnSchool of Medicine at Mount Sinai.

Characterization of Mutagenesis

Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit(Invitrogen). Specific genomic loci were amplified using Velocity DNAPolymerase (Bioline) and primers as shown in Table 2 below. Off-targetloci represent the top predicted off-target sites in the CRISPR DesignTool. For Sanger sequencing of individual alleles, primers containedappropriate overhangs for insertion between the HindIII and XhoI sitesof pcDNA3 via In-Fusion ligation-independent cloning (Clontech). PCRproducts were gel-extracted (NucleoSpin Gel and PCR Clean-up kit,Clontech), transformed into Stellar competent E. coli (Clontech), andselected on ampicillin LB agar. Individual colonies were prepped andsequenced. For deep sequencing, the gel-extracted products were pooledand further prepared for sequencing via paired-end 2×300 bp MiSeq(IIlumina) sequencing by Genewiz, Inc. Unique sequences were identifiedand quantified from merged sequenced reads. For each on-target andoff-target amplicon reference sequence, 18 bp sequences were selectedjust beyond 35 bp upstream and downstream from the 20 bp guide RNAtarget sequence. Unique sequences with exact matches to both of these 18bp sequences were extracted and collated, with an average of 170,432reads per amplicon. For each amplicon, sequences with lengths divergentfrom the reference sequence were identified as having insertions ordeletions (indels).

TABLE 2On-target and off-target genomic locations, sequences, and amplificationprimers utilized. Genomic Target + PAM Sequence Location (underlined)Primer Sequences mCherry n/a GGCCACGAGTTCGAGATCGAGGGForward: GGCGAGGAGGATAACATGG (SEQ ID NO: 5) (SEQ ID NO: 18) Reverse:CTTCAGCCTCTGCTTGATCTC (SEQ ID NO: 19) ccr5 on- chr3, +146373721CAGGTTGGACCAAGCTATGCAGG Forward: target (SEQ ID NO: 6)TTGTCATGGTCATCTGCTACTC (SEQ ID NO: 20) Reverse: GTGTCACAAGCCCACAGATATT(SEQ ID NO: 21) ccr5 off #1 chrl, −1179505884 CAGACTGGATCAAGCTATGCCAGForward: (SEQ ID NO: 7) CTCCACTTTCCATAACAGTCTAGG (SEQ ID NO: 22)Reverse: GGTCCTTGGAACAGTAGAGATAG (SEQ ID NO: 23) ccr5 off #2chr3, −132755718 CAAGTTACAACAAGCTATGCAAG Forward: (SEQ ID NO: 8)TGTTTGCTGTGAGGCTACTTTG (SEQ ID NO: 24) Reverse: TCACTGTCCAATCTGCTTTACC(SEQ ID NO: 25) ccr5 off #3 chr20, −117216675 AAGGTTTTTCCAAGCTATGCTAGForward: (SEQ ID NO: 9) GCAGAGGCATTATAAACCCAATATG (SEQ ID NO: 26)Reverse: CCAGGAGGAACTGGCAAAT (SEQ ID NO: 27) ccr5 off #4chr2, +1140172166 CAGGATTCACCAAGCTCTGCCAG Forward: (SEQ ID NO: 10)AAGCTCCATCTTCTTCGTTCTT (SEQ ID NO: 28) Reverse:AGTAGGAGATGGATTTACAGGTATT (SEQ ID NO: 29) ccr5 off #5 chr12, +159746621CAGTTTGGTTCAAGCTATGTTAG Forward: (SEQ ID NO: 11) CAGTGACATGAGCACCTGAA(SEQ ID NO: 30) Reverse: GCAAGGACATCCTCATCCATAA (SEQ ID NO: 31)efnb2 on- chr13, −1106512590 AGAATTCAGCCCTAACCTCTGGG Forward: target(SEQ ID NO: 12) CCTGGACAAGGACTGGTACTAT (SEQ ID NO: 32) Reverse:TAGCACAGGGTCCCAAATTC (SEQ ID NO: 33) efnb2 off chr7, −1136299453AGAATTCAGGCTTAACCTCTTAG Forward: #1 (SEQ ID NO: 13)GCAGGCTGGTAATTGATCTTTC (SEQ ID NO: 34) Reverse: TGATCCACAGTTGGTTGAATCC(SEQ ID NO: 35) efnb2 off chr2, +136596395 AAAATTCTTCCCTAACCTCTAAGForward: #2 (SEQ ID NO: 14) CCAGAATGTGTCCTGGGTTTAG (SEQ ID NO: 36)Reverse: GTGTCAGAGCGAGACTTTGT (SEQ ID NO: 37) efnb2 off chr7, +198992870ACATTTCAGCTCTAACCTCTGGG Forward: #3 (SEQ ID NO: 15)GGAGTATCTTCAGCTGTGAGAAG (SEQ ID NO: 38) Reverse: CTGTTACACGTTCCTTGCTACT(SEQ ID NO: 39) efnb2 off chr14, +198564083 ATAAATCAGCCCTAACATCTGAGForward: #4 (SEQ ID NO: 16) CTGATTGAGTGGGTCATCAGAA (SEQ ID NO: 40)Reverse: GCTACGTGCTGGTGCTAAA (SEQ ID NO: 41) efnb2 off chr3, −16909191AAAAGTTTGCCCTAACCTCTCAG Forward: #5 (SEQ ID NO: 17)GTCCAGGAAAGAAAGTTGCATAAG (SEQ ID NO: 42) Reverse: GCTGTCTGCTGGAAAGATAGT(SEQ ID NO: 43)

B. Results

Sendai Virus Incorporating Cas9 and a Guide RNA Flanked by Self-CleavingRibozymes Replicates to High Titer

Paramyxoviruses have a single-stranded, negative-sense RNA genome.During replication, the virus replication complex (nucleoprotein (N),phosphoprotein (P), and large RNA-dependent RNA polymerase (L)) uses thegenome as a template for production of both full length antigenome (thereverse complement of the genome) and individual capped andpolyadenylated mRNAs (FIG. 1A). The antigenome is further transcribedinto genome, thus amplifying the genome for replication. During mRNAproduction, gene start and gene stop signals within the flankingintergenic regions determine the ends of the mRNA transcript. For thisproof-of-principle study, a recombinant SeV (rSeV) with EGFP insertedbetween the N and P genes via duplication of the N-to-P intergenicregion was utilized. S. pyogenes Cas9 was inserted downstream of theEGFP reporter via a P2A ribosomal skipping sequence (FIG. 1A). Achimeric guide RNA (20 bp target sequence and 76 bp trans-activatingCRISPR RNA) was inserted as a new cassette between the P and M genes viaduplication of the P-to-M intergenic region (FIG. 1A). The guide RNA wasflanked by a self-cleaving 5′ ribozyme and a self-cleaving 3′ ribozyme,e.g. hammerhead ribozymes, to provide precise ends to the guide RNA(FIGS. 1A and 1B). The sequence of the exemplary self-cleaving 5′ribozyme and the self-cleaving 3′ ribozyme, are shown in FIG. 1B intheir conformational orientation, and in 5′ to 3′ as follows:

Rbz1: (SEQ ID NO: 2) GUGGCCCUGAUGAGCGAAACGGUAGGAAUUCCUACCGUC Rbz2:(SEQ ID NO: 3) CACCGGAGUCGACUCCGGUCUGAUGAGUCCGUGAGGACGAAAUACGU

The ribozymes were confirmed as functional for cleavage by transfectingthe DNA construct encoding the T7-driven rSeV-Cas9 positive-senseantigenome (the ribozymes are functional in the RNA positive-senseorientation) into BSR-T7 cells (BHK cells stably expressing T7polymerase). qRT-PCR on T7-transcribed antigenomic RNA extracted fromtransfected cells showed efficient self-cleavage for both ribozymes(FIG. 1C). Replication-competent rSeV-Cas9 were rescued byco-transfecting the antigenome construct with the accessory SeV-N, —P,and -L expression constructs required for genomic replication and thusvirus rescue. Tescue efficiency and/or genomic replication washypothesize as potentially being impaired or even blocked by thepresence of self-cleaving ribozymes in the antigenome. However, it wasalso hypothesized that nucleoprotein encapsidation of the antigenomicRNA would happen quickly enough to prevent formation of the ribozymestructure and thus self-cleavage of the antigenome; by contrast, mRNAsare not encapsidated, and thus the mRNA encoding the guide RNA would befree to undergo ribozyme cleavage. Unexpectedly and surprisingly,rSeV-Cas9 rescued as efficiently as a corresponding control virus withmutations in the ribozymes to prevent ribozyme activity (FIG. 1D).Although the growth kinetics of rSeV-Cas9 were slower than those of thecontrol virus, consistent with some negative effect of the ribozymes ongenomic replication, rSeV-Cas9 still reached the same peak titer ofalmost 10⁸ IU/mL (FIG. 1E), consistent with standard peak titers for SeVin cell culture. It was further confirmed that rSeV-Cas9 produced theCas9 protein upon infection. Western blot analysis of HEK293 cellseither transfected with a Cas9-expressing plasmid or infected withrSeV-Cas9 showed the expression of Cas9 protein (FIG. 1F).

rSeV-Cas9 Targeting mCherry Gene Achieves Almost Complete Mutagenesis ofa Reporter Cell Line

The initial rSeV-Cas9 incorporated a guide RNA specific for the mCherrygene (rSeV-Cas9-mCherry). A HEK293-based reporter cell line wasgenerated with inducible mCherry, and this cell line was infected at amultiplicity of infection (MOI) of 25 with either rSeV-Cas9-mCherry or acontrol virus expressing Cas9 but lacking the guide RNA cassette(rSeV-Cas9-control). Induction of mCherry expression at various dayspost-infection showed a progression of knockout over time, with knockoutappearing more pronounced starting at 4 days post-infection (FIG. 2A andFIG. 5). Quantification of this time point (induction at day 4 andcollection for flow cytometry at day 5) showed ˜80% knockout of mCherryfluorescence (FIG. 2A). Fluorescence microscopy visually confirmed thestrong reduction of mCherry fluorescence upon knockout (FIG. 2B).

The reporter cell line was utilized to confirm the requirement for theribozymes to preserve guide RNA function. Mutation of the 3′ ribozyme(rbz 2) strongly reduced reporter knockout efficiency, while mutation ofboth the 5′ and 3′ ribozymes (rbz 1/2) abrogated knockout activity (FIG.2C, compare to FIG. 2A). An alternative 3′ ribozyme was tested, thewidely-used hepatitis delta virus ribozyme, in place of the existinghammerhead ribozyme. This version of rSeV-Cas9-mCherry also efficientlyknocked out mCherry fluorescence, surprisingly with potentially evengreater efficiency (FIG. 2C).

To quantitatively assess the degree of mutagenesis induced byrSeV-Cas9-mCherry, deep sequencing was performed on the mCherry locusamplified from reporter cells collected at day 6 post-infection. 98% ofalleles had indels, indicating nearly complete mutagenesis of thereporter (FIG. 2D). These results strongly indicated that the rSeV-Cas9vector is highly efficient in targeting endogenous alleles.

rSeV-Cas9 Efficiently Mutates Endogenous Ccr5 and Efnb2

As opposed to the single allele of mCherry in the reporter cell line,there are two or more alleles of most endogenous genes per cell. To testthe ability of the Sendai virus vector to target the more abundantendogenous alleles, rSeV-Cas9 viruses were generated targeting codingexons of the human ccr5 and efnb2 genes. A preliminary test of theability of rSeV-Cas9-CCR5 was performed to induce mutagenesis resultingin functional disruption of ccr5. HEK293 cells were utilized sinceHEK293 cells express negligible levels of CCR5, which contain inducibleCD4 and CCR5 transgenes in addition to their endogenous alleles. CD4 andCCR5 are cell surface receptors required for infection by R5-tropicHIV-1, and Affinofile cells have been used extensively to characterizeCCR5-mediated HIV entry. Affinofile cells were infected withrSeV-Cas9-CCR5, and at 2 days post-infection, CD4/CCR5 overexpressionwas induced, and the cells were further infected with an R5-tropic HIV-1isolate the following day (FIG. 3A). At this early time point, cellsinfected with rSeV-Cas9-CCR5 were expected to have lower levels of CCR5relative to cells infected with rSeV-Cas9-control due to ongoingmutagenesis of the inducible ccr5 transgene and endogenous ccr5 alleles.After an additional 2 days, flow cytometry revealed efficient knockoutof the induced CCR5, and p24 staining indicative of HIV-1 infection atthe earlier time point had a 43% reduction in geometric meanfluorescence intensity compared to the rSeV-Cas9-control infection (FIG.3A).

To examine mutagenesis of endogenous alleles, HEK293 cells were infectedwith the ccr5- and efnb2-targeting rSeV-Cas9 viruses at a MOI of 25 andwere collected at 6 days post-infection. The on-target loci as well asthe top five predicted off-target sites were PCR amplified. HEK293 cellsare known to generally have 3 copies of chromosome 3 (encoding ccr5) and2-3 copies of chromosome 13 (encoding efnb2). Deep sequencing revealedhigh rates of on-target mutagenesis (75% and 88% for ccr5 and efnb2,respectively) (FIG. 3B), once again strongly suggesting that rSeV-Cas9is effective. Off-target mutagenesis was unremarkable for thisfirst-generation Cas9 without modifications to increase specificity,ranging from no detectable increase to 0.05% above the non-targetingcontrol (FIG. 3B). These results confirmed that Sendai virus delivery ofCRISPR/Cas9 can efficiently target endogenous genes.

Ccr5-Targeting rSeV-Cas9 Edits Primary Human Monocytes at High Frequency

Primary human CD14+ monocytes were infected with rSev-Cas9, which arenormally resistant to lentiviral transduction, at a MOI of 50. To bettervisualize reduction in CCR5 expression upon mutagenesis, monocytes werealso stimulated with GM-CSF to induce macrophage differentiation withconcomitant upregulation of CCR5. Cells were collected at 5 dayspost-infection, and deep sequencing revealed 88% on-target mutagenesis(FIG. 4A). Surprisingly, the two single nucleotide deletions flankingthe cleavage site together comprised 78% of all detected indels (FIG. 4Aand FIG. 6); by contrast, in HEK293 cells the same deletions togethercomprised 9% of detected indels, and no single mutation comprised morethan 10% of the total (FIG. 3B and FIG. 6). Infection of monocytes froman independent donor showed a similar result, with the above deletionscomprising ˜50% of mutant alleles (19/38 mutations via Sangersequencing), indicating that this may represent a cell type-specificphenomenon. When single specific mutations comprise such a largeproportion of the total indels, mismatch-based assays such as the T7E1endonuclease assay, which relies on highly variable mutagenesis todetect mutations, may strongly underestimate the degree of on-targetmutagenesis. As with the HEK293 cells, detected mutagenesis of predictedoff-target loci in the monocytes was negligible (FIG. 4A). Flowcytometry of infected monocytes from an independent donor confirmedknockout of cell surface CCR5 at the same time point (FIG. 4B).

Example 2: Temperature Sensitive Mutants of rSeV-Cas9 Vector

A temperature sensitive (ts) mutant of the rSev-Cas9 vector described inExample 1 above was created. This mutant was made by introducing severalmutations into the P (D433A, R434A, K437A) and L (L15581, N1197S,K1795E) genes of the recombinant Sendai virus vector, referred to hereinas the “PL mutant.” The PL mutant efficiently edits at permissivetemperatures, for example, temperatures up to and around 34° C., but iseliminated upon shifting to non-permissive temperatures, for example,temperatures above 37° C. and above. PL mutants of the Sendai virus havebeen previously reported in literature, but not for purposes ofCRISPR-Cas gene editing. Generation of PL mutants can be found at, forexample, Ban H. et al. PNAS 2011 Aug. 23; 108(34): 14234-14239, herebyincorporated by reference in its entirety. The PL mutant generated inBan H. et al. was a Z strain of Sendai virus, whereas the presentrSev-Cas9 vector used was a Fushimi F1R strain. A schematic of the tsmutations adapted from Ban H. et al. in the rSev-Cas9 vector (PL mutant)is illustrated in FIGS. 8A and 9A. The temperature sensitive phenotypeof the PL mutant is shown in FIG. 8B.

The PL mutant has been shown to be capable of infecting human CD34+hematopoietic stem cells (HSCs) from both human fetal liver andperipheral blood mobilized CD34+ HSCs at 80-90% efficiency. Furthermore,editing frequency in these HSCs at about 80%. For example, as shown inFIG. 9B, the PL mutants (ts rSeV-Cas9-CCR5 vectors) successfullyinfected and transduced purified human fetal liver CD34+ and peripheralblood mobilized CD34+ HSCs. Time course of infection is shown in FIG.9C. Sanger sequencing data, from ts rSeV-Cas9-CCR5 infected CD34+ HSCsat 2 dpi. 19/24 clones (˜80%), shown in FIG. 9D, showed indels at thetargeted CCR5 locus.

Infected CD34+ HSCs have been transplanted into immunodeficient SCID-Humice (n=9 for each group). All except one mice remained healthy at 10weeks post-transplant. This illustrated that the PL mutants did notrevert in vivo to a pathogenic virus, as wild type Sendai virus ishighly pathogenic even in wild-type immunocompetent mice, and likelywould have killed the immunodeficient SCID-Hu mice. Phenotyping ofinfected CD34+ HSCs (FIG. 10) illustrated that the PL mutants caninfect >90% of CD34+/CD38−/CD45RA−/CD90+(Thy1+)/CD49f-high cells whichare known as long-term-HSC or SCID-re-populating cells, capable ofreconstituting SCID-Hu mice at a single cell level.

Additionally, the PL mutants generated were essentially silent ininducing the IFN response compared to the non PL-mutants (i.e.non-temperature sensitive rSeV-Cas9 vectors). This is a highlysurprising but important phenotype. Furthermore, the PL mutantsgenerated in Ban H. et al. did not mention the effect (positive ornegative) of the PL mutants on the interferon response. There isadditionally no present indication why the PL mutant generated in thisExample possesses an “IFN-silent” phenotype, as Sendai virus infectionis known to induce the production of IFN. FIGS. 11A and 11B show thefold induction of 2 representative interferon stimulated genes (ISGs) in293T cells infected with either the rSeV-Cas9 vector of Example 1 or thetemperature sensitive PL mutant vector across a wide range of viralinoculum, as measured by qRT-PCR. At high viral genome copies, the PLmutant vector is markedly deficient in inducing ISGs compared to therSeV-Cas9 vector of Example 1. This remained true regardless of the gRNAcontained, either mCherry or CCR5.

Example 3: Additional Modifications to the rSeV-Cas9 Vectors

An additional deletion mutant of the rSeV-Cas9 vector described inExample 1 was created, missing the Fusion protein (i.e. ΔF mutants).These are only capable of growing in an F-complementing cell line. Thetemperature sensitive PL mutants of Example 2 are generated to be ΔFmutants. rSev-Cas9 vectors that can deliver two gRNAs are generated andillustrated in FIG. 12.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentdisclosure as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present disclosure as set forthin the claims. Such variations are not regarded as a departure from thescope of the disclosure, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference in their entireties.

1. A viral particle comprising a nucleic acid comprising a genomesequence of a single-stranded RNA (ssRNA) virus or antigenome sequencethat is complementary to the genome sequence, the antigenome sequencecomprising a first region comprising (i) a target segment; and (ii) afirst segment encoding a first self-cleaving ribozyme, wherein thetarget segment is adjacent to the first segment.
 2. The viral particleof claim 1, wherein the first region further comprises (iii) a secondsegment encoding a second self-cleaving ribozyme, wherein the targetsegment is flanked by the first segment and the second segment.
 3. Theviral particle of claim 1, wherein the first self-cleaving ribozyme is a3′ self-cleaving ribozyme or a 5′ self-cleaving ribozyme.
 4. The viralparticle of claim 3, wherein the first self-cleaving ribozyme comprisesone of a hammerhead ribozyme and a hepatitis delta virus (HDV) ribozyme.5. The viral particle of claim 1, wherein the antigenome sequencefurther comprises a second region comprising a third segment encoding anuclease.
 6. The viral particle of claim 5, wherein the second regionfurther comprises a fourth segment encoding a reporter molecule.
 7. Theviral particle of claim 5, wherein the nuclease comprises Cas9 or Cpf1.8. The viral particle of claim 1, wherein the target segment comprisesguide RNA (gRNA), wherein the gRNA has a scaffold sequence and atargeting sequence.
 9. The viral particle of claim 1, wherein the firstself-cleaving ribozyme comprises a hammerhead ribozyme.
 10. The viralparticle of claim 1, further comprising a third region comprising afifth segment, wherein the fifth segment comprises a mutant P gene. 11.The viral particle of claim 1, further comprising a fourth regioncomprising a fourth region comprising a sixth segment, wherein the sixthsegment comprises a mutant L gene.
 12. The viral particle of claim 1,wherein the viral particle is within the order mononegavirales.
 13. Theviral particle of claim 1, wherein the viral particle is within thefamily paramyxoviridae.
 14. The viral particle of claim 1, wherein theviral particle is a Sendai virus (SeV) or a Newcastle disease virus(NDV).
 15. The viral particle of claim 1, wherein the viral particle isa temperature sensitive mutant.
 16. A method of introducing into a hostcell a target RNA, comprising (i) contacting the viral particle of claim1 with said host cell; and (ii) culturing the host cell under conditionsallowing (a) producing a target RNA; and (b) liberating the target RNA,wherein the first self-cleaving ribozyme liberates the target RNA fromthe transcribed first region.
 17. The method of claim 16, wherein thehost cell is selected from the group consisting of an archaea cell,bacterial cell, and a eukaryotic cell.
 18. The method of claim 17,wherein the host cell is a stem cell.
 19. A method of introducing asite-specific modification to target DNA in a host cell comprising (i)contacting the viral particle of claim 1 with said host cell; (ii)culturing the host cell under conditions allowing (a) producing the gRNAflanked by the 5′ self-cleaving ribozyme and the nuclease; (b)liberating the gRNA, wherein the 5′ self-cleaving ribozyme liberates thegRNA; (c) expressing the nuclease; (d) forming a complex between thenuclease and the gRNA, wherein the scaffold sequence of the gRNA isbound to the nuclease; and (e) contacting the target DNA with thecomplex, wherein the targeting sequence of the gRNA binds to a sequenceon the target DNA adjacent to a protospacer adjacent motif (PAM); and(iii) introducing the site-specific modification to the target DNA. 20.The method of claim 19, wherein the site-specific modification is one ofan insertion, a deletion, a frameshift, and a point mutation.
 21. Themethod of claim 19, wherein the DNA is genomic.
 22. The method of claim19, wherein the host cell is selected from the group consisting of anarchaea cell, bacterial cell, and a eukaryotic cell.